Biochimica et Biophysica Acta 1861 (2017) 824–838

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbagen

Review

Minor snake venom proteins: Structure, function and

potential applications
Johara Boldrini-França a, Camila Takeno Cologna a, Manuela Berto Pucca b, Karla de Castro Figueiredo Bordon a,
Fernanda Gobbi Amorim a, Fernando Antonio Pino Anjolette a, Francielle Almeida Cordeiro a,
Gisele Adriano Wiezel a, Felipe Augusto Cerni a, Ernesto Lopes Pinheiro-Junior a, Priscila Yumi Tanaka Shibao a,
Isabela Gobbo Ferreira a, Isadora Sousa de Oliveira a, Iara Aimê Cardoso a, Eliane Candiani Arantes a,⁎
a
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
b
Medical School of Roraima, Federal University of Roraima, Boa Vista, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Snake venoms present a great diversity of pharmacologically active compounds that may be applied as research
Received 20 August 2016 and biotechnological tools, as well as in drug development and diagnostic tests for certain diseases. The most
Received in revised form 12 December 2016 abundant toxins have been extensively studied in the last decades and some of them have already been used
Accepted 20 December 2016
for different purposes. Nevertheless, most of the minor snake venom protein classes remain poorly explored,
Available online 22 December 2016
even presenting potential application in diverse areas. The main difficulty in studying these proteins lies on
Keywords:
the impossibility of obtaining sufficient amounts of them for a comprehensive investigation. The advent of
Snake venoms more sensitive techniques in the last few years allowed the discovery of new venom components and the in-
Toxins depth study of some already known minor proteins. This review summarizes information regarding some struc-
Drug discovery tural and functional aspects of low abundant snake venom proteins classes, such as growth factors, hyaluroni-
dases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease
inhibitors, antimicrobial peptides, among others. Some potential applications of these molecules are discussed
herein in order to encourage researchers to explore the full venom repertoire and to discover new molecules
or applications for the already known venom components.
© 2016 Published by Elsevier B.V.

1. Introduction
Snake venoms are complex mixtures of organic and inorganic com-
pounds that act on a variety of specific metabolic and physiologic targets
of preys or victims, assisting feeding and defense [1]. Protein composi-
tion of snake venoms may undergo pronounced qualitative and quanti-
tative variation in all levels of taxa, as well as in populations and
individuals of the same species [2,3]. Variation in protein expression of
venom components may also be observed in the same specimen in dif-
ferent ontogenetic stages [4]. Such great diversification in proteins that
⁎ Corresponding author at: Universidade de São Paulo, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Av. do Café s/n°,
Monte Alegre, 14040–903 Ribeirão Preto, SP, Brazil.
E-mail addresses: joharafran@gmail.com (J. Boldrini-França), camilatcbio@gmail.com
(C.T. Cologna), manupucca@hotmail.com (M.B. Pucca), karla@fcfrp.usp.br
(K.C.F. Bordon), fernandagamorim@gmail.com (F.G. Amorim), fanjolette@yahoo.com.br
(F.A.P. Anjolette), fran_acordeiro@hotmail.com (F.A. Cordeiro), gisele.wiezel@gmail.com
(G.A. Wiezel), felipe_cerni@hotmail.com (F.A. Cerni), ernesto.pinheiro@usp.br
(E.L. Pinheiro-Junior), priscila.shibao@gmail.com (P.Y.T. Shibao),
igobboferreira@yahoo.com.br (I.G. Ferreira), isadora_so@yahoo.com (I.S. de Oliveira),
iara_cardoso@hotmail.com (I.A. Cardoso), ecabraga@fcfrp.usp.br (E.C. Arantes).
compose the venom directly reflects its toxicity and pathophysiologic
effects, and may represent an evolutionary arms race, in which the
venom mixture is adapted to improve the predator's ability of subduing
different preys and to overcome the resistance of some prey species to
the venom [5,6].
Elapidae and Viperidae venom proteins are produced and secreted
by oral exocrine glands that present a basal-central lumen, where the
produced venom is stored until the moment of its delivery [7]. The pro-
duction of toxins is activated by morphological and biochemical chang-
es in secretory epithelial cells after venom injection or extraction [8–
10]. However, synthesis and secretion of different protein classes are
not synchronized and may result in variation in venom composition in
different stages of the venom production cycle [11].
Many regulatory mechanisms are supposed to impact protein com-
position in snake venoms, such as mutations affecting gene expression
[12], duplication and loss of toxin-related genes [13], post-transcrip-
tional microRNAs regulation [14] and proteolytic processing [13]. Al-
though presenting variable expression levels in different snake
venoms, the pathologically important toxins, such as phospholipases
A2, metalloproteases, kallikrein toxin families and three-finger toxins
are usually the most abundant proteins found in these biological

http://dx.doi.org/10.1016/j.bbagen.2016.12.022
0304-4165/© 2016 Published by Elsevier B.V.
 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
samples. Some of these toxins compose up to 80% of the total venom
proteins, such as phospholipase A2 from Crotalus durissus venom,
which forms the crotoxin complex that is a neurotoxin responsible for
the major clinical symptoms of crotalic envenoming in South America
[15]. The major snake toxins are encoded by large multilocus gene fam-
ilies that exhibit substantial gene duplication and directional selection
and consequently the greatest variation [13].
On the other hand, the minor snake venom proteins are supposed to
have ancillary functions, less variability and experienced little to no
gene duplication and positive selection. This contrast may be a result
of the likely conservative functions and the low relative abundance of
ancillary proteins in the venoms, which precluded their participation
in the pray-predator arm's race, resulting in a lack of selective pressures
[13,16]. Nevertheless, although some minor snake venom protein clas-
ses are supposed to present secondary effects, their ecological relevance
to the snake and their physiologic effects should be better explored. It is
also worth to mention that most of these proteins interact with specific
targets in some physiological systems, which will be discussed thereaf-
ter. Accordingly, these features have aroused an interest in investigating
possible medical and biotechnological applications for these families of
venom proteins.
Another issue to be considered is that venom production has a sub-
stantial metabolic cost [17] and a long regeneration cycle [9,18], which
indicates that the venom composition is optimized in order to maximize
efficacy while metabolic expenditure is minimized [19,20]. Thus, the
synthesis and secretion of this variety of protein classes presenting
merely ancillary and unessential functions seem contradictory consid-
ering that the maintenance of the venom repertoire is considerably
metabolic expensive. Therefore, researchers should look into minor pro-
teins classes in the venom aiming to unveil possible functions they may
play in the envenoming or in the physiology of the venom gland. More-
over, the expression of these proteins in nonvenomous tissues, the con-
servation degree of physiologic activities comparing with their related
orthologs and the structure and rate of duplication, mutations and rear-
rangement of their encoding genes may provide relevant information
regarding snake venom evolution and the generation of toxins diversity.
In spite of their possible great scientific value, some of these classes
are poorly explored mainly due to difficulties in purifying them from the
venom for an in-depth investigation [21]. In this context, this review
will focus on some classes of proteins that are known to be expressed
in relatively low amounts in most snake venoms (b 20% of the whole
venom proteins, as showed in Table 1), such as growth factors, hyal-
uronidases, cysteine-rich secretory proteins, nucleases and nucleotid-
ases, cobra venom factors, vespryns, protease inhibitors, antimicrobial
peptides, among others. Moreover, this review also aims to discuss the
importance of minor proteins to toxinology and how the advent of inno-
vative technological resources may enable the assessment of their struc-
tural and functional properties and their role in the evolutionary history
of snake venoms. We also highlight that this review represents the first
compilation of snake venom minor proteins and provides useful infor-
mation of those still neglected molecule that may hide outstanding po-
tential applications.
2. Omic approaches in the identification of minor venom proteins
Until a few years ago, comprehensive investigation of snake venoms
composition was considered a time-costing and laborious work. Mainly
due to the low sensitivity of the applied methods, usually only the major
components, such as proteases, neurotoxins and phospholipases A2,
were studied in detail [22,23]. The advances of proteomics,
transcriptomic and genomic techniques, as well as the improvement
of their related instrumentation, have led to progress in knowledge re-
garding the complexity of snake venom composition and the evolution-
ary history of those sophisticated lethal cocktails [24,25]. Not long ago,
the term “venomics” was used for the first time to describe the proteo-
mic study of snake venom composition [26,27]. However, the term has
825
evolved for a more comprehensive meaning and nowadays is also used
to describe the studies which combine strategies such as genomics,
transcriptomics and proteomics to unveil the full picture of venom com-
ponents [25,28]. A genomic analysis of the genes encoding venom pro-
teins may answer a number of fundamental questions, especially
regarding the evolutionary origin of those remarkable compounds and
the genetic basis of generating toxin function and diversity [28,29]. In
defiance of this great value, there is still a lack of snakes' full genomes
in the literature [29,30]. The big size of snakes' genomes which could
turn the assembling of novel genomes an extreme difficult task could
explain this deficiency [28]. In a recent study regarding the loss of
venom toxins genes in rattlesnakes, Dowell and colleagues [29] stated
that most investigations of snake venom diversity have been conducted
without genome sequences; besides their article, only other two groups
reported full genomes from snakes [30].
Following a different direction, transcriptomics, essentially the next
generation sequencing (NGS) transcriptomic approach, have allowed
the investigation of the venom repertoire in a broader way, revealing
transcripts that are rarely expressed or present in very low abundance
and therefore poorly studied hitherto [24,31]. Nevertheless, such strat-
egy cannot always provide accurate quantitative data nor predict the
post translational modifications (PTM), which include the cleavage of
the mature region, disulfide bonds formation, modifications of the side
chains and/or N- and C-terminal [25,32,33]. Glycosylation, as an exam-
ple, is one of the most frequent modifications found in snake venoms
and play notable role in protein folding, conformation, stability, phar-
macodynamics, and biological activity. As for other types of PTMs, glyco-
sylation increase the complexity of venom proteomes and expand
functions of its components [34], highlighting the relevance to elucidate
those protein adornments [33,35]. In parallel and to fulfill those gaps,
proteomics, supported by advances of mass spectrometry, has been
widely employed to explore animal venoms. This strategy can elucidate
not only the molecular mass of the toxin, but also fragments of its se-
quence which can be used as tags for database searches, the identifica-
tion of post translational modifications and their localization within the
sequence [36].
Individually, proteomics and transcriptomics strategies have already
contributed massively to the investigation of snake venom diversity
(Table 1). Notwithstanding, the “venomics” strategy, which is the inte-
gration of the data coming from different cutting-edge technologies,
will replace the classical approach, giving toxinologists the access be-
yond the traditional and paving the way to explore bona fide novel com-
pounds and minor venom proteins [37–39].
3. Minor venom protein classes
3.1. Growth factors
The designation “growth factor” was historically associated with
growth and cell proliferation. Later, other cellular responses were attrib-
uted to these neurotrophins, such as cell differentiation, transformation,
synthesis, secretions, death and motility [105]. The first identified
growth factor was a nerve growth factor (NGF) from mouse sarcoma
180 in the 1950s [106,107]. Snake venom NGF (sv-NGF) was firstly iso-
lated in 1956 from the venom of Agkistrodon piscivorus [108]. Some
years later, epidermal growth factor (EGF) from the submaxillary
gland of mouse and platelet-derived growth factor (PDGF) from mon-
key blood were discovered [109,110]. Since then, over 200 growth fac-
tors have been isolated and studied [105].
Among the several known families of growth factors, the following
have been identified in snakes and are deposited in UniProt (Universal
Protein Resource Knowledgebase) databank: latent-transforming
growth factor beta-binding protein (LTBP), transforming growth factor
beta-2 (TGF-β), hepatocyte growth factor-regulated tyrosine kinase
(HGF), hepatoma-derived growth factor (HDGF), delta/notch-like epi-
dermal growth factor, connective tissue growth factor, insulin-like
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Table 1
Main minor protein classes in snake venoms.

Venom protein classes Approaches Relative Main biological activities Other relevant characteristics References
used in the abundance in
identification snake venoms (%)⁎

5’-Nucleotidases T, P and T.A. b0,1 - 4,8 Hydrolysis of 5'-nucleotides to Act in synergism with ADPases, [40–42]
nucleosides, leading to platelet phospholipases, and disintegrins,
aggregation inhibition potentiating the anticoagulant effect in
the envenoming
Aminopeptidase A T, P and T.A. - Hypotension - [43–45]
Aminopeptidase N P - Hypotension - [44]
Cathelicidin-derived and
T -- Antimicrobial activity -- [46–57]
cathelicidin-related peptides
Cobra venom factors (CVFs) P, T And T.A. 0.1 – 2.8 Complement depletion Mainly found in Elapidae snakes [58–62]
Cystatins T, P and T.A. 1,7 Protease inhibition Inhibition of tumor cells metastasis [63–65]
Presence of sixteen cysteine residues
Cysteine-rich secretory proteins
P, T and TA 0,1 - 15,9 Blockage of ion channels highly conserved which form eight [66–70]
(CRISPs)
disulfide bonds
Dipeptidylpeptidase IV P and T - Alterations in the cardiovascular, - [44,45,71,72]
immune and neuroendocrine systems
and glucose hemostasis
Act as a “spreading factor” in the
Degradation of hyaluronan in
Hyaluronidases P, T and TA 0.1 – 1.9 envenoming and potentiate the effects of [73–75]
extracellular matrix
toxins
Kazal-type inhibitors P, T 8,3 Protease inhibition -- [76,77]
Diversity of biological effects, including
P, T and blockage of ion channels, disturbances in
kunitz-type inhibitors 0,3 - 12,6⁎⁎ Protease inhibition [78–87]
TA blood coagulation, fibrinolysis and
inflammation
Increases the vascular permeability and
Neuronal differentiation, synaptic
facilitates the diffusion of toxins in
Nerve growth factors (NGFs) P, T and TA 0.1 – 5 plasticity, and neuroprotection in the [88–90]
envenoming. May act as a proapoptotic
peripheral and central nervous systems.
factor
Hypotension, locomotor depression and
Phosphodiesterases (PDEs) P, T and T.A. 0.1 – 3,2 -- [91–94]
platelet aggregation
Cleavage of membrane phospholipids Hydrolysis of phospholipids at sn-1 and
Phospholipases B (PLB) P, T and TA b0.1 – 1,4 [95–97]
and hemolytic activity sn-2 positions
Vascular endothelial Proliferation of vascular endothelial cells
P, T and TA b0.1 – 3,2 -- [98–100]
growth factors (VEGFs) and hypotension
Vespryns T, P and TA 0.2 –14,4 Hypolocomotion, hypernociception Ohanin presents a single cysteine residue [101]
Structural similarity to whey acidic
Waprins T and TA -- Antimicrobial activity [102–104]
protein (WAP)

T = transcriptome; P = proteome; T.A. = traditional approach (activity-guided assays).

⁎ Relative abundance determined by proteomic approach.
⁎⁎ Exception of Dendroaspis polylepis venom that is composed by 61.1% of kunitz-type inhibitor.

growth factor-binding protein 5 (IGF), epidermal growth factor (EGF-
like protein), brain-derived neurotrophic factor (BDNF), interferon
(IF), platelet-derived growth factor (PDGF), tumor necrosis factor
(TNF), snake venom vascular endothelial growth factor (sv-VEGF) and
nerve growth factor (NGF). However, this review will focus on VEGF
and NGF, which are the most studied snake venom growth factors.
3.1.1. Nerve growth factors (NGFs)
NGFs participate in neuronal differentiation, synaptic plasticity, and
neuroprotection in the peripheral and central nervous systems [88].
NGF acts also on no neuronal cells, especially on hematopoietic stem
cells. However, most of these effects were reported for murine NGF
[111].
The physiological responses to sv-NGF have not been elucidated yet
[111]. However, the effects of mammalian NGF on non-neuronal cells
might assist the comprehension of some effects exerted by sv-NGFs on
mammalian systems [112]. It has been described that part of the sv-
NGF injected at the bite site may reach the circulation, leading to
some physiological activities on non-neuronal cells or tissues. Sv-NGF
from Naja atra (formerly N. naja atra) exerts important systemic effects
in the envenoming, such as plasma extravasation and histamine release,
which result in tissue vulnerability and facilitate toxin diffusion in the
prey organism [111]. In contrast, the sv-NGF isolated from Naja naja
has shown no toxic effects [113].
In regards to the presence of PTMs, some sv-NGFs were confirmed to
have an Asn23 glycosylation site (NXS/T). After treatment with PNGase
F, the sv-NGFs from Acanthophis antarcticus, Anilios nigrescens (formerly
Ramphotyphlops nigrescens) and Oxyuranus scutellatus were confirmed
to be N-glycosylated proteins [114]. Notably, this glycosylation site is
missing in Notechis scutatus, Pseudechis australis and Pseudechis
porphyriacus [114]. Although the sv-NGFs from the snakes Hoplocephalus
stephensii [114] and Naja sputatrix [115] have shown an N-linked glyco-
sylation site, it migrates as a non-glycosylated protein. The functional rel-
evance of these differences in PTMs for sv-NGF demands further assays.
Sv-NGF has low abundance in snake venoms, corresponding to
0.1%–0.5% (w/w) of crude venoms of Daboia russelii (formerly Vipera
russelli) [116] and of snakes from Oxyuranus [112,114], Naja [90,117,
118] and Sistrurus [119] genus. The relative abundance of ESTs coding
sv-NGF comprised only 0.1% of the total transcripts from Lachesis muta
venom gland [98].
The cDNA encoding sv-NGF exhibits a signal peptide, a pre-prodomain
and the mature protein [115]. The precursor contains a presumptive 18
amino acid signal sequence, which is followed by a propeptide (proNGF)
of approximately 109 amino acid residues [120]. The prodomain is impli-
cated to the correct folding of mature NGF [112]. The first cDNA of a sv-
NGF was reported in 2002 [121] from Bothrops jararacussu venomous
gland. The molecular model revealed that this mature sv-NGF (containing
118 amino acid residues) is formed by a pair of β-sheets, three β-hairpin
loops, a reverse turn and a short α-helix.
Concerning the functional properties and potential applications of
sv-NGFs, the injection of cobra venom NGF (cvNGF) was capable of
slow down the growth of Ehrlich ascites carcinoma (EAC) cells in
 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
mice, probably via an indirect mechanism in which tyrosine kinase A
(TrkA) receptors are involved. On the other hand, cvNGF showed prolif-
erative activity and had no cytotoxic effect on breast cancer cell line
MCF-7 [122].
Sv-NGF from N. sputatrix upregulates endogenous expression of NGF
in PC12 cells, pro-survival cell surface receptors and ion channels [115].
The neurite differentiation activity evidenced with sv-NGF from N.
sputatrix [115] and O. scutellatus [114] was similar to that of mammalian
homolog. These results may lead to further studies on sv-NGFs as poten-
tial therapeutic agents against neuronal injury.
3.1.2. Vascular endothelial growth factor (VEGF)
Vascular endothelial growth factor (VEGF), formerly designated as
vascular permeability factor, stimulates vasculogenesis, angiogenesis
or lymphangiogenesis. The VEGF family is divided into seven groups,
designated as VEGF-A to VEGF-F and placental growth factor (PGF)
[99]. Snake venom glands present at least three different VEGF-Fs with
unique features and with distinct receptor-selectivity, designated as
VEGF-F1 to VEGF-F3. Additionally, VEGF-A-like transcripts have also
been identified in some snake venoms [123]. VEGF-A is a cytokine se-
creted by tumor cells that play important role in normal and tumor-re-
lated angiogenesis.
Up to now, 26 expressed sequence tags (ESTs) for sv-VEGF were
identified by transcriptomic approach in the venom glands of
Bothropoides insularis (formerly Bothrops insularis) (19), Bothropoides
pauloensis (former Bothrops pauloensis) (1), Bothrops atrox (former
Bothrops colombiensis) (3), Crotalus durissus collilineatus (2) and L.
muta (1) snakes [98,124–126]. The sv-VEGFs corresponded to 0.1% of
the total ESTs from L. muta venom gland [98]. The VEGF gene is com-
prised of eight exons which yields the splicing isoforms VEGF-An,
where “n” may be 121, 145, 165, 183, 189 or 206 and represents the
number of amino acid residues in the protein [99]. VEGF-A165 is the
major isoform and a biological indicator of the invasiveness of hepato-
cellular carcinoma [127]. In 1999, a VEGF-like protein, named HF, was
firstly isolated from Vipera aspis aspis venom [123,128]. A cDNA
encoding for sv-VEGF was identified in B. insularis venom gland in
2001 [129]. Two sv-VEGFs (vammin and VR-1 from Vipera ammodytes
ammodytes and Daboia russelli russelli venoms, respectively) are
known to lack the C-terminal heparin-binding region found in other
heparin-binding VEGF subtypes. On the other hand, their C-terminal
recognizes similar heparin/heparan sulfate molecules and shows high
selectivity for the kinase insert domain-containing receptor (KDR)
when compared with VEGF-A165. Besides that, their C-terminal specif-
ically blocks the VEGF-A165 activity [99,130].
VEGF-F induces hypotension and proliferation of vascular endotheli-
al cells by binding to the KDR receptor [100]. VEGF-A165 and vammin
induced the synthesis and secretion of perlecan via VEGF receptor-2
(VEGFR-2) in cultured human brain microvascular endothelial cells.
That proteoglycan may help to regulate the angiogenesis, maintain the
endothelial barrier function and contribute to a synergistic effect on an-
giogenesis [131]. The activation of VEGFR-2 depends on the affinity and
concentration of the ligands and the angiogenic activity is potentiated
when the ligand binds to the co-receptor Neuropilin (Nrp). Hence,
drugs with the VEGFR-2/Nrp binding domains may be successfully de-
signed as potential proangiogenic therapies [132,133]. However, further
studies are necessary to elucidate the biological activities, mechanism of
action and possible applications of these neurotrophins from snake
venoms.
3.2. Hyaluronidases
Venom hyaluronidases are enzymes that hydrolyze preferentially
the hyaluronan, the major glycosaminoglycan found in the extracellular
matrix [73]. These enzymes belong to EC 3.2.1.35 class, which includes
the hyaluronidases found in mammals' sperm cells, lysosomes and ani-
mals venoms. They are capable of hydrolyzing glycosidic linkages
827
β → 1–4 of the residues N-acetyl-β-D-glucosamine and D-glucuronate
from hyaluronan producing tetra and hexasaccharides [74]. Therefore,
during the envenoming, hyaluronidases facilitate the venom diffusion
in the victim's tissue due to their hydrolytic characteristics, acting as a
“spreading factor” and enhancing the toxins' effects [75,134,135]. This
class of enzyme has been found in several organisms, being ubiquitous
in snake venoms [73,136,137].
Hyaluronidases from different sources have been used in different
applications in medicine. Examples include the use as a diffusion pro-
moter for active substances (drugs, for instance), the treatment of
hyaluronan-induced diseases (such as some types of cancers), and in
the aesthetic medicine [138]. Despite their great therapeutic potential,
these enzymes are found in small proportions in snake venoms and
they have an extremely unstable catalytic activity. These issues hamper
their isolation from venoms, as well as their in-depth functional and
structural characterization [139,140]. This is reinforced by the few hyal-
uronidase amino acids sequences deposited in databases, which are cur-
rently fourteen for Uniprot and seventeen for NCBI.
The first report indicating the occurrence of hyaluronidase in snake
venoms was in 1939 by Duran-Reynalds, who analyzed the hyaluroni-
dase activity in the venom of nine different snake species [134]. Howev-
er, up to date, limited studies have successfully isolated these enzymes
from snake venoms. Reports include the hyaluronidases from
Deinagkistrodon acutus (formerly Agkistrodon acutus) [141], Agkistrodon
contortrix [142], N. naja [136], D. russelli [143], Cerastes cerastes [135],
Crotalus durissus terrificus [137] and Lachesis muta rhombeata [140].
Snake venom hyaluronidases are glycoproteins with molecular mass
ranging from 28 to 70 kDa, with optimum enzyme activity detected
around pH 5.5, 37 °C and 0.15–0.2 M NaCl. They hold specificity for
hyaluronan and show no significant activity upon chondroitin sulfate
[135,136,140–143]. Moreover, hyaluronidase activity can vary accord-
ing to the snake's age [144], species [145,146] and habitat [147,148].
The enzyme activity is inhibited by divalent cations, high temperatures
and salt concentrations, extreme pH and by EDTA, heparin, denaturing
agents and herbal extracts [135,137,142,143].
As previously mentioned, in vivo experiments have proven that
snake venom hyaluronidase acts as a spreading factor, enhancing the ef-
fect of venom toxins [135–137,143]. Therefore, many recent studies put
a spotlight on the production of antibodies and on the search for syn-
thetic and natural inhibitors of these enzymes in order to improve anti-
venom therapy, since the importance of hyaluronidases in the
envenoming process is outstanding [135,136,149–155].
3.3. Cysteine-rich secretory proteins
Cysteine-rich secretory proteins (CRISPs) are non-enzymatic com-
ponents present in various organisms [66]. They are also found in
snake venoms, but their function in envenoming has not been fully un-
derstood thus far [156]. Snake venom CRISPs are single chain proteins
with molecular masses ranging from 20 to 30 kDa. They display sixteen
highly conserved cysteine residues that form eight disulfide bonds [66].
CRISPs are distributed among Viperidae and Elapidae families from
different continents. There are reports of the isolation and cloning of
three snake venom CRISPs from Agkistrodon piscivorus piscivous,
Ophiophagus hannah and Crotalus atrox, named piscivorin, ophanin
and catrin, respectively [157]. Other CRISPs such as triflin, ablomin,
latisemin and tigrin were isolated from the venoms of Protobothrops
flavoviridis, Agkistrodon blomhoffi, Laticauda semifasciata and Rhabdophis
tigrinus tigrinus, respectively. Ablomin, triflin and latisemin block the
caffeine-induced depolarization, but they have no effects on the con-
traction of the smooth muscle, indicating their L-type Ca2 + channel
blocker action. On the other hand, tigrin does not inhibit depolarization
and contraction induced by caffeine [67]. Pseudechetoxin (PsTx) and
pseudecin, from P. australis and P. porphyriacus venoms, respectively,
block the cyclic nucleotide-gated (CNG) ion channel [67,68].
828 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
Natrin, from N. a. atra venom, was reported to be a BKCa channel
blocker [69]. Its three-dimensional structure, determined by X-ray crys-
tallography, revealed three functional sites: a pathogenesis-related pro-
tein from the group 1 (PR-1) like domain localized at the N-terminal
region, a highly flexible cysteine-rich domain (CRD) at the C-terminal
extremity of the protein, and a hinge between these domains. The
CRD domain plays an important role in blocking the ion channel activity
since it probably interacts with the pore of Kv1.3 channels by hydrogen
bonds formation [70].
Besides blocking the ion channel activity, CRISPs may act as inflamma-
tory modulators [158] once they can regulate the expression of adhesion
molecules in endothelial cells. Lecht et al. reported that a CRISP from Echis
carinatus sochureki negatively regulates angiogenesis, thus disturbing the
wound healing process of envenomed victims [159]. Furthermore,
crovirin, a CRISP purified from Crotalus viridis viridis [160] was tested in
vitro for its antiparasitic activity. This CRISP demonstrated a dose-depen-
dent antiparasitic activity on Leishmania amazonensis and Trypanosoma
cruzi amastigote forms and Trypanosoma brucei rhodesiense metacyclic
and blood trypomastigotes form.
In the light of the above, CRISPs are a promising class of snake venom
component that can be employed as a model for future cancer drugs due
to the negative regulation of angiogenesis. In addition, this class of com-
ponents may assist the development of new drugs against leishmaniosis
and Chagas disease.
3.4. Nucleases and nucleotidases
Nucleases and nucleotidases are hydrolytic enzymes ubiquitously dis-
tributed in snake venoms. Their main function is related to the generation
of adenosine through their catalytic activity, as shown in Fig. 1, which
schematically illustrates adenosine production by these enzymes. Adeno-
sine may support toxin biodistribution and also contributes to prey
immobilization, whereas it increases vascular permeability, inhibits neu-
rotransmitter release and promotes hypotension, sedation, locomotor de-
pression and bradycardia. Although nucleases and nucleotidases activities
are broadly detected in snake venoms, the biological effects of these en-
zymes are not completely clear [45,71,161–163].
3.4.1. Nucleases
Nucleases are hydrolytic enzymes that cleave nucleic acids and their
derivatives [91]. They are classified as endonucleases and exonucleases.
Endonucleases [164] comprise DNAses [165], and RNAses [166] which
Fig. 1. Schematic representation of adenosine production/generation by snake venom toxins. During the envenoming, toxins (in green) promote cellular lysis, releasing nucleic acids (DNA
and RNA). Nucleic acids become susceptible to the action of snake venom nucleases, resulting in the production of 5′-nucleotides, which are cleaved by snake venom phosphatases,
generating adenosine. Another pathway to produce adenosine involves the cleavage of ATP by ATPase or phosphodiesterase, producing ADP and AMP, which are degraded by
nucleotidases, yielding adenosine.
hydrolyze DNA and RNA, respectively. Exonucleases comprise phospho-
diesterases (PDEs) [94] that catalyze the hydrolysis of phosphodiester
bonds, releasing 5′-mononucleotides [91].
Endonucleases show optimum activity on acidic pH and do not re-
quire divalent cations for nucleic acids hydrolysis. On the other hand,
PDEs have optimum activity on basic pH and divalent metal ions are re-
quired for PDE's hydrolytic activity [167–169].
Endonucleases and PDEs have been found in snake venoms from
Elapidae family and crotaline and viperine snakes from Viperidae family
[71]. The biological activities of endonucleases have not been elucidated
yet [91] Nevertheless, PDEs are known to lead to hypotension, locomo-
tor depression [92] and inhibition of platelet aggregation [93].
In general, nucleases can lead to the production of purine and
pyrimidine nucleotides that might act synergistically with nucleotidases
and others snake venom toxins (phospholipases A2, cardiotoxins,
myotoxins, cytolytic peptides and proteases/hemorrhagins) [71,91,
161,170–173], contributing to the symptomatology and overall pathol-
ogy caused by the snake bite envenoming [37].
3.4.2. Nucleotidases
Nucleotidases are enzymes involved in the cleavage of nucleic acid
derivatives and nucleic acid-related substrates (ATP, ADP and AMP).
However, specific identification of snake venom nucleotidases is im-
paired by the presence of other toxins that share similar substrate spec-
ificity [174].
Nucleotidases have been found in a great number of snake venoms
[168], but there are only few nucleotidases isolated and characterized
so far due to their minor amounts in venoms [174,175]. The biological
activities of nucleotidases are known to be involved in the digestive pro-
cess and in endogenous purines release, potentiating the venom-in-
duced hypotension and paralysis via purine receptors. However,
nucleotidases activities need to be deeper investigated [71,161]. Fur-
thermore, some reports in the literature show that nucleotidases can
act independently or in synergism with other toxins, like phosphodies-
terase, phosphomonoesterase, endonuclease, phospholipases A2, cyto-
toxins, myotoxins, and heparinases [42,71,161,176–178].
Snake venom nucleotidases comprise 5′-nucleotidase, ATPase and
ADPase. 5′-Nucleotidase are metalloenzymes of high molecular mass
[42,168,179,180], ranging from 73 to 100 kDa [42,177,181,182]. Gulland
and Jackson [183] firstly identified the 5′-nucleotidase activity in snake
venoms. These enzymes selectively hydrolyze 5′-nucleotides to nucleo-
sides [180] and inhibit platelet aggregation by adenosine release [40–
 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
42]. Thus, 5′-nucleotidase increases the anticoagulant effect of ADPases,
phospholipases A2 and disintegrins by acting synergistically with these
toxins [184].
ATPases were firstly described by Zeller in 1950 and are known to
hydrolyze ATP, releasing adenosine and pyrophosphate. Depending on
the reaction conditions, ATPases can cleave ATP into AMP and pyro-
phosphate or into ADP and phosphate. ATPases have not been isolated
from snake venoms yet, and their biological activity remains unclear.
However, they may be involved in shock symptoms by depletion of
ATP [185], since ATP released from skeletal muscles cells (due to activity
of myotoxins) is quickly hydrolyzed by ATPase, and act as a danger sig-
nal molecule stimulating purinergic receptors, contributing to the path-
ophysiology of envenomation [186–188].
ADPase hydrolyzes ADP, producing adenosine and orthophosphate
[168,189]. The only ADPase from snake venom isolated so far has
94 kDa and it was purified from D. acutus. This toxin inhibits platelet ag-
gregation in platelet rich plasma induced by different molecules (ADP,
collagen and sodium arachidonate), but it does not inhibit thrombin-in-
duced aggregation in platelet poor plasma [177]. The inhibition of plate-
let aggregation is also attributed to adenosine release due to ADPase
activity [174].
3.5. Protease inhibitors
Snake venoms are interesting sources of protease inhibitors, al-
though these molecules represent, in most cases, only a small propor-
tion of snake venoms. The control of most body's chemical reactions is
sustained by the antagonism of: (i) proteases, which play key functions
in different systems and biochemical pathways, and (ii) the correspon-
dent protease inhibitors, responsible for controlling protease activity
[190]. In this context, the possible function of protease inhibitors in
snake venoms is to promote an imbalance in prey homeostasis, since
these proteins can inhibit some classes of enzymes, like serine proteases
[191,192], acetylcholinesterase (AchE) [193] as well as interact with ion
channels [194,195], thus interfering in the physiological systems medi-
ated by these components. On the other hand, it was suggested that
these protease inhibitors may also assist the proteolysis control while
the venom is stored at the venom gland [79,196].
The first protease inhibitor found in snake venom was reported in
1972 when Takahashi and colleagues [197] isolated a potent kunitz-
type protease inhibitor from the venom of D. russelli. The occurrence
of this family of protease inhibitors is reported in Elapidae and Viperidae
snake venoms [79,198–200].
This class of protease inhibitors presents a conserved motif found in
kunitz bovine pancreatic trypsin inhibitor, which is the active domain
responsible for the inhibition of proteases such as trypsin, chymotryp-
sin, elastase, thrombin, and activated factor X [201,202]. Structurally,
they display approximately sixty amino acid residues, with six con-
served cysteine residues involved in three disulfide bonds, supporting
the stable and compact structure of the folded peptide [203–205].
These venom components represent from 0.3% to 4.6% of total pro-
teins in snake venoms [198,206–208]. The exceptions identified thus
far are the species Dendroaspis polylepis and D. angusticeps, whose
venom proteome presented larger amounts of kunitz type protease in-
hibitors [163]. Although these inhibitors may exhibit several structural
similarities, they show an extensive range of biological functions, in-
cluding blockage of ion channels, disturbances in blood coagulation, in-
flammation and fibrinolysis [78–81].
A kunitz-type protease inhibitor isolated from Macrovipera lebetina
venom displayed a remarkable anti-tumor activity by the interaction
with an integrin receptor [82]. In a further study, this molecule inhibited
angiogenesis and endothelial cell adhesion and migration, as well as in-
creased microtubule dynamics. Therefore, this toxin is able to interfere
in crucial mechanisms related to the process of tumorigenesis [83].
Some studies also proposed an antibleeding function for these toxins,
since some kunitz-type snake venom protease inhibitors are capable
829
of inhibiting enzymes involved in the blood coagulation [84–87]. Thus,
due to their extensive biological roles, these venom proteins may pres-
ent promising biotechnological and pharmaceutical applications.
Other minor classes of protease inhibitors found in snake venoms
are the cysteine protease inhibitors (cystatins), the metalloproteases in-
hibitors and the kazal-type protease inhibitors. The latter was only re-
ported in Bothriechis ssp. venom, with a relative abundance of 8.3 and
9% in B. schlegelii and B. supraciliaris species, respectively [76,77]. Re-
cently, a kazal-type inhibitor-like protein that is able to inhibit trypsin
activity at pH 5.4 was purified from B. schlegelii venom. This finding sug-
gests that such protein may act controlling venom proteolysis, consider-
ing that the venom gland has an acidic pH [196].
Metalloprotease inhibitors were found by proteomic approaches in
snake venoms from Echis ocellatus, C. c. cerastes [209] and V. anatolica
[206]. Recently, a new group of metalloprotease peptide inhibitors
(Poly-Gly- and poly-His-poly-Gly-peptides and tripeptides) was detect-
ed in snake venoms. Tripeptides were identified in the venom of
Bothrops ayerbei [210], Bothriechis supraciliaris [77] and Bothrocophias
campbelli [211]. Interestingly, these inhibitors are encoded by the
same precursor of bradykinin potentiating and C-natriuretic peptides.
Furthermore, metalloprotease inhibitors such as Poly-His-poly-Gly-
peptides (pHpG peptides) were identified in the venoms of Bothrops
snakes [212,213] and E. ocellatus [209], whereas poly-Gly-peptides (pG
peptides) were found in the venom of Bothriechis supraciliaris [77].
Regarding snake venom cystatins, the first toxin of this class was iso-
lated from Vipera ammodytes by Kregar and colleagues, in 1981 [214].
The only proteomic study that determined the percentage of this class
of toxin in snake venom revealed that it represents 1.8% and 9.8% of
total venom proteins of the species Bitis gabonica gabonica and Bitis
arietans, respectively [198]. Moreover, three other cystatins were puri-
fied from snake venom, all of them belonging to type-2 cystatins,
which present about 120 amino acid residues and two characteristic di-
sulfide loops [215–217]. Nonetheless, no studies have elucidated their
role in the envenoming process yet.
Recently, the pharmaceutical application of a recombinant snake
venom cystatin (sv-cystatin) in cancer therapy was investigated. The re-
combinant cystatin is capable of inhibiting the invasion and metastasis
of tumor cells, both in in vitro and in vivo assays [64]. Furthermore, it
was reported that this molecule is capable of inhibiting tumor angiogen-
esis and tumor-endothelial cell adhesion [63,65]. Additionally, a
recombinant adenovirus transfected with the sv-cystatin gene has dem-
onstrated the ability to inhibit the invasion and metastasis of several
types of tumors, such as mouse melanoma cells, human gastric carcino-
ma cells, and MHCC97H cells. This recombinant adenovirus approach
demonstrated an increased antitumor activity compared to the recom-
binant protein itself [63].
Protease inhibitors are becoming molecules of interest to biotechno-
logical and pharmaceutical areas since proteases are one of the main
potential drug targets. There are about 553 human gene products incor-
porating protease domains. In addition, these enzymes are also found in
bacteria, virus and parasites [218,219]. In this context, the search for
new protease inhibitors plays a key role to warrant the discovery of
more effective drugs for human therapeutics, especially for cancer
treatment.
3.6. Antimicrobial peptides
Antimicrobial peptides are found in several organisms and are part of
their innate-immune system, participating of host defense and contrib-
uting to the survival of these organisms in microbe-rich environments
[220,221]. Snake venoms contain biologically active peptides, which
are a rich source of possible novel antimicrobials agents [222]. Further-
more, several well-characterized snake toxins, such as phospholipases
A2, metalloproteases, L-amino acid oxidases and crotamine have shown
effects against many microorganisms [223,224].
830 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
Most of the studies regarding antimicrobial peptides from snake
venoms report only the screening of crude venom antimicrobial
activity [225–228]. These peptides are mainly identified by mass spec-
trometry following chromatographic isolation, although genomic se-
quencing and functional characterization by different antimicrobial
assays are also employed [46,47,104,220]. Among the previously de-
scribed snake venom antimicrobial peptides, it is possible to highlight
cathelicidins, viperidins, waprins, β-defensins, beyond other not yet
classified molecules.
One of the major AMP families in mammals is composed by
cathelicidin peptides that are cationic peptides with highly heteroge-
neous structures [229,230]. These peptides are composed of 12 to 80
amino acid residues which are mainly folded in α-helical structures [230].
The cathelicidin-BF, a cathelicidin-derived antimicrobial peptide,
was the first cathelicidin peptide identified in reptiles (Bungarus
fasciatus snake) [48,49] and presents in vitro antimicrobial activity on
bacterial and fungal species [48]. Other cathelicidin-derived peptides
were identified in O. hannah (OH-CATH and its derivatives OH-
CATH30 and OH-CM6) [46,47,50,51], B. fasciatus (BF-CATH and its de-
rivatives BF-30 and BF-15) [48,52–54], Hydrophis cyanocinctus (Hc-
CATH) [55] and N. atra (NA-CATH) [46,56]. These peptides exhibit anti-
microbial activity against gram-positive and gram-negative bacterial
strains and fungi. Furthermore, it presents antitumor activity, as dem-
onstrated against BF-CATH cells [48].
Another family of snake venom AMPs is the vipericidins (cathelicidin-
related peptides). This family comprises toxins from South American pit
vipers [57], such as crotalicidin (C. d. terrificus), lachesicidin (L. m.
rhombeata), batroxicidin (Bothrops atrox) and lutzicidin (Bothrops lutzi).
These venom-derived peptides showed in vitro antimicrobial activity
against several bacterial strains, including Streptococcus pyogenes, Staphy-
lococcus aureus, Escherichia coli, Klebsiella pneumonia, Enterococcus faecalis,
Acinetobacter baumannii and Pseudomonas aeruginosa [57].
Omwaprin, a cationic antimicrobial peptide belonging to the waprin
family, was isolated from O. microlepidotus snake venom [104]. This anti-
microbial peptide is composed of 50 amino acid residues and contains a
whey acid protein (WAP) domain in its structure (PDB ID. 3NGG).
Omwaprin features in vitro antimicrobial activity against Bacillus
megaterium and Staphylococcus warnei gram-positive bacterial strains
[104]. On the other hand, the occurrence of waprins in the venoms from
Thrasops jacksonii, Liophis poecilogyrus, Philodryas olfersii, Pseudoferania
polylepis (formerly Enhydris polylepis and Rhabdophis tigrinus was report-
ed only through transcriptomic approaches [102]. Corrêa-Netto et al.
[103] reported that waprin-like peptides correspond to 0.2% of the total
toxins in the cDNA library of Micrurus altirostris venom gland.
The peptide Pep5Bj (1.37 kDa), isolated from Bothropoides jararaca
venom, was able to inhibit the growth of yeast (Candida albicans) and
phytopathogenic fungi (Fusarium oxysporum and Colletotrichum
lindemuthianum) cell cultures [231]. Another peptide of 2.49 kDa,
known as NAP (Naja Antibacterial Peptide), was isolated from Indian
cobra (N. naja) venom and showed in vitro potent antibacterial activity
against gram-negative and gram-positive bacterial strains [232].
Phylogenetic analysis enabled the identification of 13 β-defensin-
like sequences from Bothrops spp. and Lachesis spp. snakes by PCR ap-
proach [233]. β-Defensins-like peptides have also been described in
other snake venoms [234,235] and display distinct biological effects, in-
cluding antimicrobial activity [236].
These snake venom antimicrobial peptides can be valuable to the de-
velopment of novel antimicrobial drugs [47,48,222,237], besides their
potential as biomarkers as well as diagnostic tools [238,239].
3.7. Phospholipases B
Phospholipases are enzymes that catalyze the cleavage of phospho-
lipids, releasing fatty acids and lysophospholipids. Depending on the
class of phospholipase, this reaction may take place in four different
sites in membrane phospholipids. When catalyzed by phospholipase B
(PLB), the cleavage occurs at sn-1 and sn-2 positions [95]. PLBs were poor-
ly reported so far since they are rarely found in snake venoms, in contrast
to phospholipases A2 (PLA2s) that are amongst the major venom
components.
There are several snake species that have PLB activity in their venom,
such as Pseudechis porphyriacus, P. australis, N. naja, D. russelii, Cerastes
vipera and Crotalus adamanteus [240–242]. PLBs were firstly reported
in Pseudechis colletti venom as a dimeric protein of 16.5 kDa that pre-
sents high hemolytic activity in erythrocytes and cytotoxicity to rhabdo-
myosarcoma cells [96].
The omics era not only facilitated but also fastened the discovery of
novel PLBs. The proteomic analysis of the Australian snake Pseudechis
guttatus revealed two peptide sequences that shared identity to PLB.
They were identified in one basic spot of 2D–PAGE and these two pep-
tides covered 5% of the PLB sequence. The authors suggest that this
basic toxin may contribute to the hemolysis observed in P. guttatus
envenoming [97].
Recently, proteins with PLB sequence identity were found in Micrurus
dumerilii snake venom proteome, although in small relative abundance
(b 0.1%). This venom is mainly composed of PLA2 and three-finger toxins
[243]. Interestingly, one PLB was identified by mass spectrometry analysis
in the venom of the Brazilian snake L. m. rhombeata. It was the first report
of this class of protein in Lachesis genus [140].
Although PLBs have been reported in some snake venom proteomes,
the advent of transcriptome techniques has allowed the identification of
this class in different snake genus. One EST that encodes a PLB from a
new lipase family was identified in the cDNA library of Drysdalia
coronoides venom gland. This EST comprises a signal peptide of 36
amino acid residues and a mature protein of around 60 kDa. Moreover,
the authors also found some peptides in D. coronoides venom proteome
that correspond to this protein [244].
Rokyta and colleagues [245] analyzed the transcriptome from C.
adamanteus venom gland and found one transcript that encodes a PLB
precursor presenting a signal peptide of 36 amino acid residues and
the mature PLB of 526 amino acid residues. It represents just 0.06% of
the whole transcriptome.
Furthermore, the P. flavoviridis venom gland transcriptome revealed
that PLB comprises 0.14% of the transcripts, while in Ovophis okinavensis
the percentage of sequences that encodes this protein was just 0.15%.
Some peptide sequences corresponding to PLB were also found in the
proteome of these two snake venoms and they cover from 26.1 to
61.6% of the PLB sequence [45].
The venom-gland transcriptome of the coral snake Micrurus fulvius
revealed one PLB sequence, representing 0.154% of total reads. This
venom is mostly composed of PLA2 (64.9% of toxins reads), which is re-
sponsible for the neurotoxic, cardiotoxic, myotoxic, anticoagulant and
hemolytic effects of the envenoming [246].
Vonk and colleagues [16] analyzed the genome from the venom and
accessory glands of king cobra snake (O. hannah) and observed that they
are mainly composed of three-finger toxins (venom) and lectins (acces-
sory gland). In addition to these findings, they also reported PLB tran-
scripts in these two glands. In contrast, PLB could not be found in the
venom proteome of this snake.
Although some PLBs have been identified in snake venoms by geno-
mic, transcriptomic and proteomic approaches, they are poorly studied
by traditional approach, mainly due to their low abundance in crude
venoms [242]. Therefore the importance of omics approaches to the dis-
closure of minor components such as PLB is reinforced and further
structural and functional studies regarding this neglected class of
venom protein are required.
3.8. Cobra venom factor
Cobra venom factor (CVF) is a family of proteins that activates the
complement system. The CVF function was reported by [58]. However,
before that, Flexner and Noguchi [247] had already demonstrated that
 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
Naja genus venom produces hemolysis, which is dependent of the com-
plement system, when incubated with mammalian erythrocytes and
serum [248]. In 1971, it was shown that the venom of N. haje (Egyptian
cobra) selectively consumes the complement components C3 → C9
[249]. Lately, several studies focused on CVF purification and complete
functional characterization [250–253]. Thereby, CVF has been studied
for more than a century.
CVF presents approximately 149 kDa and shares high identity de-
gree with human complement component C3 (50% of identity, 69% of
similarity) [62]. The C3 is the most important protein of the comple-
ment system, being activated by all the three pathways: classical, lectin
and alternative, besides their possible cross-reactivity [59]. The C3 acti-
vation and the generation of its products lead to all biological functions
of the complement system [254]. Since CVF is a C3-like protein, after the
cleavage of factor B (by factor D), it is turned into CVFBb, which acts as a
C3-convertase. The continuous action of CVFBb is responsible for com-
plement depletion and production of components of the terminal
phase, including the cleavage of C5 and the generation of C5a. The C5a
component is a powerful pro-inflammatory peptide. Along with C3a,
C5a could be an important product for the snake since they increase
the vascular permeability and blood flow in the bite site, allowing the
rapid diffusion of the toxic venom compounds into the blood stream.
Excellent detailed reviews can be found elsewhere [62,255].
So far, CVF is mainly present in Elapidae snake family and it has been
described in four different genera: Hemachatus, Naja, Ophiophagus and
Austrelaps [60–62,249]. Nevertheless, complement depletion caused
by CVF is most studied using the venoms of N. naja, Naja kaouthia and
N. atra, even being found in low amounts in these venoms. Laustsen et
al. [162] demonstrated, by proteomic analysis, that CVF accounts for
0.1% of N. kaouthia whole venom, which is a very low percentage in
comparison to other components, such as PLA2 (13.5%), neurotoxins
(53.2%) and LAAO (0.4%). However, recently, CVF was also found in
the Viperidae snake (Bothrops jararaca) using transcriptomic [256]
and proteopeptidomic [257] analysis.
Based on the low relative abundance of CVF in snake venoms and its
important anti-complement effect, molecular biology emerges as a con-
venient approach to CVF production for therapeutic purposes. In this
way, recombinant [62,258] and humanized [259] CVF are available
nowadays.
3.9. Vespryn
Vespryn is a new family of venom proteins comprising ohanin from
O. hannah [101], Thai cobrin (P82885) from N. kaouthia [260] and
ohanin-like proteins from N. n. atra [261], L. muta [98] and Tropidechis
carinatus [262]. However, only ohanin (the first family member) has
been well characterized so far.
Ohanin was firstly purified in 2005 from the crude venom of O.
hannah (king cobra). It presents a molecular mass of approximately
12 kDa, with 107 amino acid residues and a single cysteine residue,
which is a unique feature of this protein class. The amino acid sequence
of ohanin shares 93% of sequence identity with Thai cobrin isolated from
N. kaouthia [101].
Ohanin induces hypolocomotion and hypernociception in mice after
intraperitoneal injection in a dose-dependent manner. However, it is
considered non-toxic since the analysis of gross pathology, after 24 h
of its injection in mice, showed no signs of hemorrhage or necrosis in
the brain, heart, lungs, kidneys, spleen and liver. Thus, the ohanin effects
are possibly important to slow down the mobility of the prey, assisting
in its capture [101].
The complete cDNA and genomic organization of ohanin was al-
ready reported. The ohanin-encoding cDNA (1558 bp) show that this
toxin is synthesized as a prepro-protein in the venom gland [263]. In
this context, due to ohanin low recovery from the venom (0.1%), heter-
ologous expression of this protein has already been accomplished and
the resulting recombinant form functionally resembles the native
831
toxin. Recombinant ohanin may be helpful in the investigation of the
structure-function relationship of this class of toxins [101].
3.10. Dipeptidylpeptidase IV
Dipeptidylpeptidase IV (DPP IV) presents a wide distribution among
snake venoms [264]. It has been identified by proteome and tran-
scriptome approaches, for example, in Rhinocerophis alternatus (former-
ly Bothrops alternatus), Gloydius brevicaudus, P. flavoviridis and O.
okinavensis venoms [44,45,72] and its activity have been detected in a
great variety of snake venoms [264,265]. The DPP IV G. brevicaudus
was partially isolated from this snake venom and the cDNA sequence
was cloned from the venom gland cDNA library. Two DPP IV, named
DPP IVa and DPP IVb, containing 751 amino acid residues were obtain-
ed. They contain a consensus sequence similar to serine proteases
around the Ser616 and a putative catalytic triad (Ser616, Asp694 and
His76) is located at the C-terminus region. The mature protein present-
ed 116 kDa estimated by SDS-PAGE, and about 25% of their molecular
mass is due to glycosylation. In fact, the sequences present 10 potential
N-glycosylation sites [265]. It is interesting that DPP IV is secreted into
the venom gland through exosome-like vesicles not through the com-
mon pathway by secretory granules and exocytosis [266].
The role of DPP IV during envenoming is still not clear, but these en-
zymes might contribute to alterations in the cardiovascular system
leading to hypotension by cleaving endogenous hypertensive factors.
Additionally, DPP IV may also present important roles in the immune
and neuroendocrine systems and in the glucose homeostasis [71].
3.11. Aminopeptidases
Aminopeptidases have been identified by “omics” techniques in the
venoms of B. jararaca [43], O. okinavensis [45], P. flavoviridis [45] and G.
brevicaudus [44]. Aminopeptidase activity was detected in the mice
envenoming by C. d. terrificus venom [267]. Furthermore, aminopepti-
dase A (APA) is released to the venom gland lumen through exosome-
like vesicles, the same say as DPP IV [266].
The rhiminopeptidase A is an APA isolated from Bitis rhinoceros
which is an exo-metallopeptidase that removes acidic terminal residues
from proteins and peptides at presence of calcium. No specificity for
acidic, basic or neutral pH was detected in the absence of calcium. Its
molecular mass was estimated by SDS-PAGE being 150 kDa, and its se-
quence is composed of 951 amino acid residues [268].
The aminopeptidases mammalian A and N act by different ways in
the renin-angiotensin system. Aminopeptidase N (APN) catalyzes the
conversion of angiotensin III to angiotensin IV through the hydrolysis
of the N-terminal arginine from angiotensin III thus preventing hyper-
tension. In contrast, APA induces hypotension by degrading angiotensin
II to angiotensin III through the hydrolysis of the N-terminal aspartic
acid since angiotensin III is less hypertensive that angiotensin II at pe-
ripheral sites [45]. Both angiotensins (II and III) also take part in the
brain renin-angiotensin system, involved in the blood pressure control
and both present hypertensive action with affinity to the AT1 and AT2
receptors [269].
Besides that, a leucine aminopeptidase was also identified and it
might be linked to the action of hemorrhagic snake venom
metalloproteases, other venom peptidases and the L-amino acid ox-
idase activity [45].
3.12. Other minor snake venom toxins
Table 2 summarizes the main minor snake venom toxins identified
only by “omic” approaches.
3.12.1. Cytokine-like molecules
Transcriptomic analyses have enabled the identification of cytokine-
like molecules in the venoms of D. acutus, R. alternatus and B. gabonica.
832 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838

Table 2
Snake venom proteins identified by “omic” techniques.

Protein class Snake venoms Approach used Possible function in the venom References
in the identification

Bungarus fasciatus, Naja atra,

Cathelicidins Transcriptome Antimicrobial activity [46,48]
Ophiophagus hannah
Deinagkistrodon acutus, Rhinocerophis Local inflammation and alterations in the
Cytokine-like molecules Transcriptome [72,270,271]
alternatus, Bitis gabonica vascular system
Crotalus adamanteus, Bothropoides
EF-hand protein Transcriptome and Proteome Unknown [276–278]
insularis
Insulin-like growth factor Ophiophagus hannah Transcriptome and Proteome Unknown [16,61,208]
Lysosomal acid lipase (LAL) Echis coloratus Transcriptome and Proteome Unknown [103,279]
Bothropoides jararaca, Bothrops fonsecai,
Metalloprotease inhibitors
Bothrops cotiara, Bothrops atrox, Bothrops
(poly-Gly- and poly-His- Proteome and Peptidome Inhibition of metalloprotease activity [77,209–213]
barnetti, Bothrops ayerbei, Bothriechis
poly-Gly-peptides and tripeptides)
supraciliaris, Bothrocophias campbelli
Neuroprotective protein Deinagkistrodon acutus Transcriptome Unknown [271]
Increased vascular permeability,
hypotension, inhibition of
Nucleosides Dendroaspis polylepis Proteome neurotransmitter release, sedation, [45,71,161–163]
locomotor depression, bradycardia,
influence in the toxin distribution
Transforming growth factor Deinagkistrodon acutus Transcriptome Unknown [271]
Sistrurus catenatus edwardsii, Echis
Renin-like aspartic protease Transcriptome and Proteome Local hypertension [280,281]
ocellatus
Transferrin-like proteins (TLP) Pseudechis australis Proteome Antimicrobial activity [282]
Deinagkistrodon acutus, Rhinocerophis
Tumor necrosis factor Transcriptome Local inflammation [72,271]
alternatus
Cerberus rynchops, Micrurus corallinus, Platelet aggregation induction and
Veficolins Transcriptome [103,274,275]
Philodryas olfersii interference in the fibrin formation

Their role in the envenoming remains unknown, but they possibly act in
the vascular system, contributing to the local inflammatory process in-
duced by the venom [72,270,271].
3.12.2. Veficolins
Ficolins are a group of proteins similar to complement-activating
lectins present in different tissues. They show a collagen-like domain
in the N-terminal and a fibrinogen-like domain in the C-terminal region
and are related to the innate defense system [272–274]. Veficolins
(venom ficolins) were identified for the first time as a venom toxin in
the venom gland transcriptome of the colubrid snake Cerberus rynchops
[274], although Ching et al. [275] reported a veficolin from P. olfersii as a
putative protein in its venom. The collagen-like domain of ryncolins
(the C. rynchops ficolins) may induce platelet aggregation while the di-
merization of two C-terminal fibrinogen-like domains might interfere in
fibrin formation by mimicking the C-terminal globular domain of fibrin-
ogen. Veficolins were also identified in the transcriptome of Micrurus
corallinus [103,274,275].
3.12.3. EF-hand protein
EF-hand proteins present a conserved calcium-binding domain,
composed of two alpha helixes linked by a loop (helix-loop-helix) usu-
ally formed by 12 amino acid residues. This helix-loop-helix motif that
coordinates calcium-binding resembles the human hand thumb and
forefinger, which provided the name EF-hand for this motif. This do-
main is important to calcium-dependent processes, such as membrane
fusion and vesicular transport [276]. Calglandulin, an EF-hand protein
identified in the B. insularis venom gland, has been implicated in the se-
cretion of toxins from the venom gland cells [245,276,283]. On the other
hand, reticulocalbin 2 EF-hand calcium-binding domain precursor is a
highly expressed nontoxin in the venom gland C. adamanteus [245,
277]. However, it is not yet clear if this molecule present toxic properties
and potential role in the envenoming or if it presence in the venom pro-
teome is just a result of the leakage of a housekeeping protein.
4. Conclusions and perspectives
Snake venoms are rich combinations of bioactive compounds that
act on specific biochemical and physiological targets of the prey or vic-
tim, causing an imbalance on their homeostasis. Thus, snake venoms are
considered a source of harmful toxins, possessing a diversity of mole-
cules that mimic some natural regulatory components [1,284,285]. On
the other hand, snake venoms toxins are also considered promising
molecules for the treatment and diagnosis of certain pathologies, as
well as for research and biotechnological purpose [284 286–288]. Fig.
2 summarizes the main potential application of the minor venom pro-
tein classes discussed herein. The most abundant toxins are easily iden-
tified and purified from snake venoms by classical approaches. They
have been extensively studied in the last decades and some of them
have already been directly applied in the treatment of some disorders
or used as models for drug designs, such as serine proteases, disintegrins
and bradykinin-potentiating peptides [288]. A classic example is Cap-
topril, which is an orally available peptidomimetic of a bradykinin
potentiating peptide from B. jararaca venom. This molecule compet-
itively inhibits the angiotensin-converting enzyme (ACE) and is ex-
tensively used worldwide in the treatment of human hypertension
[289]. Currently, nine snake venom protein derivatives are commer-
cially available for therapeutic application or under clinical trials
[288], but none of them are related to the protein classes approached
in this review.
Most of the minor snake venom proteins remained unidentified or
poorly explored for many years, mainly due to the difficulties in
obtaining them in sufficient amounts for a comprehensive study [21].
Only with the advent of new highly sensitive methods of molecular
identification, such as the “omic” approaches, this hidden world of
snake toxins could be brought to light. However, an in-depth investiga-
tion of the biological effects and the potential for therapeutic and bio-
technological application of these toxins is still necessary.
Heterologous protein expression and peptide synthesis represent al-
ternatives to produce toxins in both laboratory and industrial scale,
which may enable the study of minor snake toxin classes and generate
 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
Fig. 2. Minor snake venom protein classes and their main potential applications. The summary of the minor protein classes discussed herein are illustrated. Magenta: antimicrobial class.
Green: proteases inhibitors class. Yellow: growth factors class. Blue: other classes. The possible applications are numbered and indicated in the right panel, accordingly.
perspectives for their further investigation and application [21]. Further-
more, we highlight the importance of continuing the search for novel
toxin scaffolds, classes and isoforms, in order to enrich our knowledge
on these natural libraries of pharmacologically active molecules. Improv-
ing the understanding in snake proteins diversity is indispensable not
only to drug discovery, but also to better comprehend the pathophysiolo-
gy of snake bites and, thereby, improving the still outdated antivenom
therapy.
Minor snake venom protein classes may also provide important infor-
mation regarding the evolutionary processes that generated snake venom
repertories. Snake toxins are supposed to have been originated by genes
that were expressed both on venom gland and other tissues, followed
by tissue-specific enhanced expression in the venom gland and reduction
of expression or gene loss in nonvenomous-related tissues [256,290,291].
Evidences that support this hypothesis is the expression of various venom
homolog genes in nonvenom gland-related tissues of venomous and non-
venomous snakes [291]. Subsequently, the main toxic and abundant
snake venom proteins probably suffered selective pressures and
underwent diversification by gene duplication that occurs via “birth and
death” model [285], followed by mutations in key residues or domains
in the molecular surface [16,292] and/or gene recombination [29]. This
process typically results in multi-gene families, with conserved few disul-
fide-rich molecular scaffolds, but encoding a variety of proteins exhibiting
diverse biological and toxic activities [6,293,294]. The reason for generat-
ing toxin diversification in the venom is still not completely clarified. Nev-
ertheless, it may represent a selective advantage to these animals since
different toxins may act on synergism, enhancing toxic effects, and may
extend molecular targets in preys [294,295].
On the other hand, genes encoding many low abundant snake
venom proteins were presumably devoid of selective pressures, mainly
due to their small contribution in the prey capture [13]. Thus, the minor
protein families discussed herein are expected to present low degree of
diversification and neofunctionalization, retaining most of the biological
833
effects of their homologous from nonvenomous tissues. However, stud-
ies related to the origin of these protein classes in the venom are recent
and still very scarce and inconclusive. The analysis of the differences in
the expression rates of these proteins in the venom gland and in other
tissues and the comparative characterization of venom proteins and
their homologs may indicate whether protein may have evolved to dis-
play toxic effects or another role in these complexes biological. More-
over, investigations related to the gene structure and expression of
venom proteins will assist the understanding of the evolutionary pro-
cesses that originated venom components and enlightening of the se-
lective forces that have been driven adaptation of snake venoms to
different ecological niches and preys.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgments
The authors acknowledge the financial support from the Fundação
de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo Re-
search Foundation, grant n. 2011/23236-4; scholarships to: GAW, n.
2014/06170-8; KCFB, n. 2013/26619-7; JBF, n. 2015/16714-8; PYTS n.
2014/15644-3; CTC, n. 2013/26200-6; FAC, n. 2012/13590-8; MBP, n.
2012/12954-6 and FGA, n. 2011/12317-3), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq, The National Council
for Scientific and Technological Development, 303689/2013-7; scholar-
ship to FAC n. 140949/2015-1), Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES, Coordination for the Improvement of
Higher Education Personnel, scholarship to GAW) (scholarship to ELPJ,
ISO and IGF) and the Support Nucleus for Research on Animal Toxins
(NAP-TOXAN-USP, grant n. 12–125432.1.3).
834 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
References
[1] B.G. Fry, K. Roelants, D.E. Champagne, H. Scheib, J.D.A. Tyndall, G.F. King, T.J.
Nevalainen, J.A. Norman, R.J. Lewis, R.S. Norton, C. Renjifo, R.C.R. de la Vega, The
toxicogenomic multiverse: Convergent recruitment of proteins into animal
venoms, Annu. Rev. Genomics Hum. Genet. 10 (2009) 483–511.
[2] N.R. Casewell, Venom evolution: Gene loss shapes phenotypic adaptation, Curr.
Biol. 26 (2016) R849–R851.
[3] J.P. Chippaux, V. Williams, J. White, Snake venom variability: methods of study, re-
sults and interpretation, Toxicon 29 (1991) 1279–1303.
[4] J.J. Calvete, L. Sanz, P. Cid, P. de la Torre, M. Flores-Diaz, M.C. Dos Santos, A. Borges,
A. Bremo, Y. Angulo, B. Lomonte, A. Alape-Giron, J.M. Gutierrez, Snake venomics of
the Central American rattlesnake Crotalus simus and the South American Crotalus
durissus complex points to neurotoxicity as an adaptive paedomorphic trend
along Crotalus dispersal in South America, J. Proteome Res. 9 (2010) 528–544.
[5] J.J. Calvete, L. Sanz, Y. Angulo, B. Lomonte, J.M. Gutierrez, Venoms, venomics,
antivenomics, FEBS Lett. 583 (2009) 1736–1743.
[6] D. Kordis, F. Gubensek, Molecular evolution of Bov-B LINEs in vertebrates, Gene
238 (1999) 171–178.
[7] H.I. Rosenberg, Histology, histochemistry, and emptying mechanism of the venom
glands of some elapid snakes, J. Morphol. 123 (1967) 133–155.
[8] S.M. Carneiro, V.R. Pinto, C. Jared, L.A. Lula, F.P. Faria, A. Sesso, Morphometric stud-
ies on venom secretory cells from Bothrops jararacussu (Jararacuçu) before and
after venom extraction, Toxicon 29 (1991) 569–580.
[9] U. Oron, A. Bdolah, Regulation of protein synthesis in the venom gland of viperid
snakes, J. Cell Biol. 56 (1973) 177–190.
[10] E. Kochva, Oral glands of the Reptilia, Biology of the Reptilia, 8, 1978 43–161.
[11] M.S. Luna, R.H. Valente, J. Perales, M.L. Vieira, N. Yamanouye, Activation of Bothrops
jararaca snake venom gland and venom production: A proteomic approach, J. Pro-
teome 94 (2013) 460–472.
[12] D.R. Rokyta, K.P. Wray, J.J. McGivem, M.J. Margres, The transcriptomic and proteo-
mic basis for the evolution of a novel venom phenotype within the timber rattle-
snake (Crotalus horridus), Toxicon 98 (2015) 34–48.
[13] N.R. Casewell, S.C. Wagstaff, W. Wuster, D.A.N. Cook, F.M.S. Bolton, S.I. King, D. Pla,
L. Sanz, J.J. Calvete, R.A. Harrison, Medically important differences in snake venom
composition are dictated by distinct postgenomic mechanisms, Proc. Natl. Acad.
Sci. U. S. A. 111 (2014) 9205–9210.
[14] J. Durban, A. Perez, L. Sanz, A. Gomez, F. Bonilla, S. Rodriguez, D. Chacon, M. Sasa, Y.
Angulo, J.M. Gutierrez, J.J. Calvete, Integrated "omics" profiling indicates that
miRNAs are modulators of the ontogenetic venom composition shift in the Central
American rattlesnake, Crotalus simus simus, BMC Genomics 14 (2013).
[15] J. Boldrini-Franca, C. Correa-Netto, M.M.S. Silva, R.S. Rodrigues, P. De La Torre, A.
Perez, A.M. Soares, R.B. Zingali, R.A. Nogueira, V.M. Rodrigues, L. Sanz, J.J. Calvete,
Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: As-
sessment of geographic variation and its implication on snakebite management, J.
Proteome 73 (2010) 1758–1776.
[16] F.J. Vonk, N.R. Casewell, C.V. Henkel, A.M. Heimberg, H.J. Jansen, R.J. McCleary, H.M.
Kerkkamp, R.A. Vos, I. Guerreiro, J.J. Calvete, W. Wüster, A.E. Woods, J.M. Logan,
R.A. Harrison, T.A. Castoe, A.P. de Koning, D.D. Pollock, M. Yandell, D. Calderon, C.
Renjifo, R.B. Currier, D. Salgado, D. Pla, L. Sanz, A.S. Hyder, J.M. Ribeiro, J.W.
Arntzen, G.E. van den Thillart, M. Boetzer, W. Pirovano, R.P. Dirks, H.P. Spaink, D.
Duboule, E. McGlinn, R.M. Kini, M.K. Richardson, The king cobra genome reveals
dynamic gene evolution and adaptation in the snake venom system, Proc. Natl.
Acad. Sci. U. S. A. 110 (2013) 20651–20656.
[17] M.D. McCue, Cost of producing venom in three North American pitviper species,
Copeia (2006) 818–825.
[18] D. Rotenberg, E.S. Bamberger, E. Kochva, Studies on ribonucleic acid synthesis in
the venom glands of Vipera palaestinae (Ophidia, Reptilia), Biochem. J. 121
(1971) 609–612.
[19] W.K. Hayes, The snake venom-metering controversy: levels of analysis, assump-
tions, and evidence, The biology of rattlesnakes 2008, pp. 191–220.
[20] D. Morgenstern, G.F. King, The venom optimization hypothesis revisited, Toxicon
63 (2013) 120–128.
[21] J. Boldrini-Franca, R. Santos Rodrigues, L.K. Santos-Silva, D.L. de Souza, M.S. Gomes,
C.T. Cologna, E. de Pauw, L. Quinton, F. Henrique-Silva, V. de Melo Rodrigues, E.C.
Arantes, Expression of a new serine protease from Crotalus durissus collilineatus
venom in Pichia pastoris and functional comparison with the native enzyme,
Appl. Microbiol. Biotechnol. 99 (2015) 9971–9986.
[22] J.J. Calvete, Venomics, what else? Toxicon 60 (2012) 427–433.
[23] Q. Kaas, D.J. Craik, Bioinformatics-aided venomics, Toxins (Basel) 7 (2015)
2159–2187.
[24] R.K. Brahma, R.J. McCleary, R.M. Kini, R. Doley, Venom gland transcriptomics for
identifying, cataloging, and characterizing venom proteins in snakes, Toxicon 93
(2015) 1–10.
[25] K. Sunagar, D. Morgenstern, A.M. Reitzel, Y. Moran, Ecological venomics: How ge-
nomics, transcriptomics and proteomics can shed new light on the ecology and
evolution of venom, J. Proteome 135 (2016) 62–72.
[26] A. Bazaa, N. Marrakchi, M. El Ayeb, L. Sanz, J.J. Calvete, Snake venomics: compara-
tive analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes,
Cerastes vipera and Macrovipera lebetina, Proteomics 5 (2005) 4223–4235.
[27] P. Juarez, L. Sanz, J.J. Calvete, Snake venomics: characterization of protein families
in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tan-
dem mass spectrometry analysis, Proteomics 4 (2004) 327–338.
[28] A. Menez, R. Stocklin, D. Mebs, 'Venomics' or : The venomous systems genome pro-
ject, Toxicon 47 (2006) 255–259.
[29] N.L. Dowell, M.W. Giorgianni, V.A. Kassner, J.E. Selegue, E.E. Sanchez, S.B. Carroll,
The deep origin and recent loss of venom toxin genes in rattlesnakes, Curr. Biol.
26 (2016) 2434–2445.
[30] H.M. Kerkkamp, R.M. Kini, A.S. Pospelov, F.J. Vonk, C.V. Henkel, M.K. Richardson,
Snake Genome Sequencing: Results and Future Prospects, Toxins (Basel) 8 (2016).
[31] A. Zelanis, A.K. Tashima, Unraveling snake venom complexity with 'omics' ap-
proaches: challenges and perspectives, Toxicon 87 (2014) 131–134.
[32] S.G. Conticello, Y. Gilad, N. Avidan, E. Ben-Asher, Z. Levy, M. Fainzilber, Mechanisms
for evolving hypervariability: the case of conopeptides, Mol. Biol. Evol. 18 (2001)
120–131.
[33] S. Dutertre, A.H. Jin, Q. Kaas, A. Jones, P.F. Alewood, R.J. Lewis, Deep venomics re-
veals the mechanism for expanded peptide diversity in cone snail venom, Mol.
Cell. Proteomics 12 (2013) 312–329.
[34] D. Andrade-Silva, A. Zelanis, E.S. Kitano, I.L.M. Junqueira-de-Azevedo, M.S. Reis, A.S.
Lopes, S.M.T. Serrano, Proteomic and glycoproteomic profilings reveal that post-
translational modifications of toxins contribute to venom phenotype in snakes, J.
Proteome Res. 15 (2016) 2658–2675.
[35] J.R. Prashanth, R.J. Lewis, S. Dutertre, Towards an integrated venomics approach for
accelerated conopeptide discovery, Toxicon 60 (2012) 470–477.
[36] P. Favreau, L. Menin, S. Michalet, F. Perret, O. Cheneval, M. Stocklin, P. Bulet, R.
Stocklin, Mass spectrometry strategies for venom mapping and peptide sequenc-
ing from crude venoms: case applications with single arthropod specimen, Toxicon
47 (2006) 676–687.
[37] J.W. Fox, A brief review of the scientific history of several lesser-known snake
venom proteins: L-amino acid oxidases, hyaluronidases and phosphodiesterases,
Toxicon 62 (2013) 75–82.
[38] J.J. Calvete, B. Lomonte, A bright future for integrative venomics, Toxicon 107
(2015) 159–162.
[39] P.F. Campos, D. Andrade-Silva, A. Zelanis, A.F. Paes Leme, M.M. Rocha, M.C.
Menezes, S.M. Serrano, I.L. Junqueira-de-Azevedo, Trends in the evolution of
snake toxins underscored by an integrative omics approach to profile the venom
of the colubrid Phalotris mertensi, Genome Biol Evol (2016).
[40] X. Chen, X.D. Yu, M. Deng, H. Li, Q.Y. He, J.P. Liu, Purification and characterization of
5'-nucleotidase from Trimeresurus albolabris venom, Zool. Res. 29 (2008) 399–404.
[41] M.L. Hart, D. Kohler, T. Eckle, D. Kloor, G.L. Stahl, H.K. Eltzschig, Direct treatment of
mouse or human blood with soluble 5'-nucleotidase inhibits platelet aggregation,
Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1477–1483.
[42] C. Ouyang, T.F. Huang, Inhibition of platelet aggregation by 5′-nucleotidase purified
from Trimeresurus gramineus snake venom, Toxicon 21 (1983) 491–501.
[43] G.S. Dias, E.S. Kitano, A.H. Pagotto, S.S. Sant'anna, M.M. Rocha, A. Zelanis, S.M.
Serrano, Individual variability in the venom proteome of juvenile Bothrops jararaca
specimens, J. Proteome Res. 12 (2013) 4585–4598.
[44] J.F. Gao, Y.F. Qu, X.Q. Zhang, Y. He, X. Ji, Neonate-to-adult transition of snake
venomics in the short-tailed pit viper, Gloydius brevicaudus, J. Proteome 84
(2013) 148–157.
[45] S.D. Aird, Y. Watanabe, A. Villar-Briones, M.C. Roy, K. Terada, A.S. Mikheyev, Quan-
titative high-throughput profiling of snake venom gland transcriptomes and
proteomes (Ovophis okinavensis and Protobothrops flavoviridis), BMC Genomics
14 (2013) 790.
[46] H. Zhao, T.X. Gan, X.D. Liu, Y. Jin, W.H. Lee, J.H. Shen, Y. Zhang, Identification and
characterization of novel reptile cathelicidins from elapid snakes, Peptides 29
(2008) 1685–1691.
[47] S.A. Li, W.H. Lee, Y. Zhang, Efficacy of OH-CATH30 and its analogs against drug-re-
sistant bacteria in vitro and in mouse models, Antimicrob. Agents Chemother. 56
(2012) 3309–3317.
[48] Y. Wang, J. Hong, X. Liu, H. Yang, R. Liu, J. Wu, A. Wang, D. Lin, R. Lai, Snake
cathelicidin from Bungarus fasciatus is a potent peptide antibiotics, PLoS One 3
(2008) e3217.
[49] Y. Wang, Z. Zhang, L. Chen, H. Guang, Z. Li, H. Yang, J. Li, D. You, H. Yu, R. Lai,
Cathelicidin-BF, a snake cathelicidin-derived antimicrobial peptide, could be an ex-
cellent therapeutic agent for Acne vulgaris, PLoS One 6 (2011) e22120.
[50] Y. Zhang, H. Zhao, G.Y. Yu, X.D. Liu, J.H. Shen, W.H. Lee, Y. Zhang, Structure-function
relationship of king cobra cathelicidin, Peptides 31 (2010) 1488–1493.
[51] B.Y. Zhang, S.M. Li, Z.H. Gao, J.H. Shen, Protective effects of snake venom antimicro-
bial peptide OH-CATH on E. coli induced rabbit urinary tract infection models, Zool.
Res. 34 (2013) 25–32.
[52] W. Chen, B. Yang, H. Zhou, L. Sun, J. Dou, H. Qian, W. Huang, Y. Mei, J. Han, Struc-
ture-activity relationships of a snake cathelicidin-related peptide, BF-15, Peptides
32 (2011) 2497–2503.
[53] H. Wang, M. Ke, Y. Tian, J. Wang, B. Li, Y. Wang, J. Dou, C. Zhou, BF-30 selectively
inhibits melanoma cell proliferation via cytoplasmic membrane permeabilization
and DNA-binding in vitro and in B16F10-bearing mice, Eur. J. Pharmacol. 707
(2013) 1–10.
[54] H. Zhou, J. Dou, J. Wang, L. Chen, H. Wang, W. Zhou, Y. Li, C. Zhou, The antibacterial
activity of BF-30 in vitro and in infected burned rats is through interference with
cytoplasmic membrane integrity, Peptides 32 (2011) 1131–1138.
[55] L. Wei, J. Gao, S. Zhang, S. Wu, Z. Xie, G. Ling, Y.Q. Kuang, Y. Yang, H. Yu, Y. Wang,
Identification and characterization of the first cathelicidin from sea snakes with
potent antimicrobial and anti-inflammatory activity and special mechanism, J.
Biol. Chem. 290 (2015) 16633–16652.
[56] R.J. Blower, S.M. Barksdale, M.L. van Hoek, Snake cathelicidin NA-CATH and smaller
helical antimicrobial peptides are effective against Burkholderia thailandensis, PLoS
Negl. Trop. Dis. 9 (2015) e0003862.
[57] C.B. Falcao, B.G. de La Torre, C. Perez-Peinado, A.E. Barron, D. Andreu, G. Radis-
Baptista, Vipericidins: a novel family of cathelicidin-related peptides from the
venom gland of South American pit vipers, Amino Acids 46 (2014) 2561–2571.
 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
[58] C.B. Ewing, The action of rattlesnake venom upon the bactericidal power of the
blood serum, Boston Med. Surg. J. 130 (1894) 487–490.
[59] A. Laich, R.B. Sim, Complement C4bC2 complex formation: an investigation by sur-
face plasmon resonance, Biochim. Biophys. Acta 1544 (2001) 96–112.
[60] S. Rehana, R. Manjunatha Kini, Molecular isoforms of cobra venom factor-like pro-
teins in the venom of Austrelaps superbus, Toxicon 50 (2007) 32–52.
[61] C.H. Tan, K.Y. Tan, S.Y. Fung, N.H. Tan, Venom-gland transcriptome and venom pro-
teome of the Malaysian king cobra (Ophiophagus hannah), BMC Genomics 16
(2015) 687.
[62] C.W. Vogel, D.C. Fritzinger, Cobra venom factor: Structure, function, and humaniza-
tion for therapeutic complement depletion, Toxicon 56 (2010) 1198–1222.
[63] Q. Xie, N. Tang, Y. Lin, X. Wang, X. Lin, J. Lin, Recombinant adenovirus snake venom
cystatin inhibits the growth, invasion, and metastasis of B16F10 cells in vitro and in
vivo, Melanoma Res. 23 (2013) 444–451.
[64] Q. Xie, N. Tang, R. Wan, Y. Qi, X. Lin, J. Lin, Recombinant snake venom cystatin in-
hibits the growth, invasion and metastasis of B16F10 cells and MHCC97H cells in
vitro and in vivo, Toxicon 57 (2011) 704–711.
[65] Q. Xie, N. Tang, R. Wan, Y. Qi, X. Lin, J. Lin, Recombinant snake venom cystatin in-
hibits tumor angiogenesis in vitro and in vivo associated with downregulation of
VEGF-A165, Flt-1 and bFGF, Anti Cancer Agents Med. Chem. 13 (2013) 663–671.
[66] Y. Yamazaki, T. Morita, Structure and function of snake venom cysteine-rich secre-
tory proteins, Toxicon 44 (2004) 227–231.
[67] Y. Yamazaki, H. Koike, Y. Sugiyama, K. Motoyoshi, T. Wada, S. Hishinuma, M. Mita,
T. Morita, Cloning and characterization of novel snake venom proteins that block
smooth muscle contraction, Eur. J. Biochem. 269 (2002) 2708–2715.
[68] R.L. Brown, L.L. Lynch, T.L. Haley, R. Arsanjani, Pseudechetoxin binds to the pore
turret of cyclic nucleotide-gated ion channels, J. Gen. Physiol. 122 (2003) 749–760.
[69] J. Wang, B. Shen, M. Guo, X. Lou, Y. Duan, X.P. Cheng, M. Teng, L. Niu, Q. Liu, Q.
Huang, Q. Hao, Blocking effect and crystal structure of natrin toxin, a cysteine-
rich secretory protein from Naja atra venom that targets the BKCa channel, Bio-
chemistry 44 (2005) 10145–10152.
[70] F. Wang, H. Li, M.N. Liu, H. Song, H.M. Han, Q.L. Wang, C.C. Yin, Y.C. Zhou, Z. Qi, Y.Y.
Shu, Z.J. Lin, T. Jiang, Structural and functional analysis of natrin, a venom protein
that targets various ion channels, Biochem. Biophys. Res. Commun. 351 (2006)
443–448.
[71] S.D. Aird, Ophidian envenomation strategies and the role of purines, Toxicon 40
(2002) 335–393.
[72] K.C. Cardoso, M.J. Da Silva, G.G. Costa, T.T. Torres, L.E. Del Bem, R.O. Vidal, M.
Menossi, S. Hyslop, A transcriptomic analysis of gene expression in the venom
gland of the snake Bothrops alternatus (urutu), BMC Genomics 11 (2010) 605.
[73] K.C.F. Bordon, G.A. Wiezel, F.G. Amorim, E.C. Arantes, Arthropod venom Hyaluron-
idases: biochemical properties and potential applications in medicine and biotech-
nology, J Venom Anim Toxins incl Trop Dis 21 (2015).
[74] S.P. Mackessy, Handbook of venoms and toxins of reptiles, Taylor & Francis, 2010.
[75] S. Pukrittayakamee, D.A. Warrell, V. Desakorn, A.J. Mcmichael, N.J. White, D.
Bunnag, The hyaluronidase activities of some Southeast Asian snake venoms,
Toxicon 26 (1988) 629–637.
[76] B. Lomonte, J. Escolano, J. Fernandez, L. Sanz, Y. Angulo, J.M. Gutierrez, J.J. Calvete,
Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis
lateralis and Bothriechis schlegelii, J. Proteome Res. 7 (2008) 2445–2457.
[77] B. Lomonte, W.C. Tsai, F. Bonilla, A. Solorzano, G. Solano, Y. Angulo, J.M. Gutierrez,
J.J. Calvete, Snake venomics and toxicological profiling of the arboreal pitviper
Bothriechis supraciliaris from Costa Rica, Toxicon 59 (2012) 592–599.
[78] W. Chen, L.P. Carvalho, M.Y. Chan, R.M. Kini, T.S. Kang, Fasxiator, a novel factor XIa
inhibitor from snake venom, and its site-specific mutagenesis to improve potency
and selectivity, J. Thromb. Haemost. 13 (2015) 248–261.
[79] S.T.H. Earl, R. Richards, L.A. Johnson, S. Flight, S. Anderson, A. Liao, J. de Jersey, P.P.
Masci, M.F. Lavin, Identification and characterisation of kunitz-type plasma kalli-
krein inhibitors unique to Oxyuranus sp. snake venoms, Biochimie 94 (2012)
365–373.
[80] C.T. Guo, S. McClean, C. Shaw, P.F. Rao, M.Y. Ye, A.J. Bjourson, Trypsin and chymo-
trypsin inhibitor peptides from the venom of Chinese Daboia russellii siamensis,
Toxicon 63 (2013) 154–164.
[81] A.K. Mukherjee, S.P. Mackessy, S. Dutta, Characterization of a kunitz-type protease
inhibitor peptide (Rusvikunin) purified from Daboia russelii russelii venom, Int. J.
Biol. Macromol. 67 (2014) 154–162.
[82] M. Morjen, O. Kallech-Ziri, A. Bazaa, H. Othman, K. Mabrouk, R. Zouari-Kessentini,
L. Sanz, J.J. Calvete, N. Srairi-Abid, M. El Ayeb, J. Luis, N. Marrakchi, PIVL, a new ser-
ine protease inhibitor from Macrovipera lebetina transmediterranea venom, im-
pairs motility of human glioblastoma cells, Matrix Biol. 32 (2013) 52–62.
[83] M. Morjen, S. Honore, A. Bazaa, Z. Abdelkafi-Koubaa, A. Ellafi, K. Mabrouk, H.
Kovacic, M. El Ayeb, N. Marrakchi, J. Luis, PIVL, a snake venom kunitz-type serine
protease inhibitor, inhibits in vitro and in vivo angiogenesis, Microvasc. Res. 95
(2014) 149–156.
[84] S.M. Flight, L.A. Johnson, Q.S. Du, R.L. Warner, M. Trabi, P.J. Gaffney, M.F. Lavin, J. de
Jersey, P.P. Masci, Textilinin-1, an alternative anti-bleeding agent to aprotinin: Im-
portance of plasmin inhibition in controlling blood loss, Br. J. Haematol. 145 (2009)
207–211.
[85] P.P. Masci, A.N. Whitaker, L.G. Sparrow, J. de Jersey, D.J. Winzor, D.J. Watters, M.F.
Lavin, P.J. Gaffney, Textilinins from Pseudonaja textilis textilis. Characterization of
two plasmin inhibitors that reduce bleeding in an animal model, Blood Coagul. Fi-
brinolysis 11 (2000) 385–393.
[86] E.K. Millers, M. Trabi, P.P. Masci, M.F. Lavin, J. de Jersey, L.W. Guddat, Crystal struc-
ture of textilinin-1, a kunitz-type serine protease inhibitor from the venom of the
Australian common brown snake (Pseudonaja textilis), FEBS J. 276 (2009)
3163–3175.
835
[87] J.C. Perez, E.E. Sanchez, Natural protease inhibitors to hemorrhagins in snake
venoms and their potential use in medicine, Toxicon 37 (1999) 703–728.
[88] R.J. Mannion, M. Costigan, I. Decosterd, F. Amaya, Q.P. Ma, J.C. Holstege, R.R. Ji, A.
Acheson, R.M. Lindsay, G.A. Wilkinson, C.J. Woolf, Neurotrophins: peripherally
and centrally acting modulators of tactile stimulus-induced inflammatory pain hy-
persensitivity, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 9385–9390.
[89] K. Sunagar, B.G. Fry, T.N. Jackson, N.R. Casewell, E.A. Undheim, N. Vidal, S.A. Ali, G.F.
King, K. Vasudevan, V. Vasconcelos, A. Antunes, Molecular evolution of vertebrate
neurotrophins: co-option of the highly conserved nerve growth factor gene into
the advanced snake venom arsenal, PLoS One 8 (2013) e81827.
[90] T. Kostiza, C.A. Dahinden, S. Rihs, U. Otten, J. Meier, Nerve growth factor from the
venom of the Chinese cobra Naja naja atra: purification and description of non-
neuronal activities, Toxicon 33 (1995) 1249–1261.
[91] B.L. Dhananjaya, D.S. CJ, An overview on nucleases (DNase, RNase, and phosphodi-
esterase) in snake venoms, Biochemistry (Mosc) 75 (2010) 1–6.
[92] F.E. Russell, F.W. Buess, J. Strassbe, Zootoxicological properties of venom phospho-
diesterase, Toxicon 1 (1963) 99–108.
[93] M.L. Santoro, T.S. Vaquero, A.F. Paes Leme, S.M. Serrano, NPP-BJ, a nucleotide
pyrophosphatase/phosphodiesterase from Bothrops jararaca snake venom, inhibits
platelet aggregation, Toxicon 54 (2009) 499–512.
[94] S. Uzawa, Über die phosphomonoesterase und die phosphodiesterase, J. Biochem.
15 (1932) 1–10.
[95] A. Aloulou, Y.B. Ali, S. Bezzine, Y. Gargouri, M.H. Gelb, Phospholipases: an overview,
Methods Mol. Biol. 861 (2012) 63–85.
[96] A.W. Bernheimer, R. Linder, S.A. Weinstein, K.S. Kim, Isolation and characterization
of a phospholipase B from venom of Collett's snake, Pseudechis colletti, Toxicon 25
(1987) 547–554.
[97] V.L. Viala, D. Hildebrand, M. Trusch, R.K. Arni, D.C. Pimenta, H. Schluter, C. Betzel,
P.J. Spencer, Pseudechis guttatus venom proteome: Insights into evolution and
toxin clustering, J. Proteome 110 (2014) 32–44.
[98] I.L. Junqueira-de-Azevedo, A.T. Ching, E. Carvalho, F. Faria, M.Y. Nishiyama Jr., P.L.
Ho, M.R. Diniz, Lachesis muta (Viperidae) cDNAs reveal diverging pit viper mole-
cules and scaffolds typical of cobra (Elapidae) venoms: implications for snake
toxin repertoire evolution, Genetics 173 (2006) 877–889.
[99] Z.K. Otrock, J.A. Makarem, A.I. Shamseddine, Vascular endothelial growth factor
family of ligands and receptors: review, Blood Cells Mol. Dis. 38 (2007) 258–268.
[100] Y. Yamazaki, K. Takani, H. Atoda, T. Morita, Snake venom vascular endothelial
growth factors (VEGFs) exhibit potent activity through their specific recognition
of KDR (VEGF receptor 2), J. Biol. Chem. 278 (2003) 51985–51988.
[101] Y.F. Pung, P.T. Wong, P.P. Kumar, W.C. Hodgson, R.M. Kini, Ohanin, a novel protein
from king cobra venom, induces hypolocomotion and hyperalgesia in mice, J. Biol.
Chem. 280 (2005) 13137–13147.
[102] B.G. Fry, H. Scheib, L. van der Weerd, B. Young, J. McNaughtan, S.F. Ramjan, N. Vidal,
R.E. Poelmann, J.A. Norman, Evolution of an arsenal: structural and functional di-
versification of the venom system in the advanced snakes (Caenophidia), Mol.
Cell. Proteomics 7 (2008) 215–246.
[103] C. Corrêa-Netto, I.e.L. Junqueira-de-Azevedo, D.A. Silva, P.L. Ho, M. Leitão-de-
Araújo, M.L. Alves, L. Sanz, D. Foguel, R.B. Zingali, J.J. Calvete, Snake venomics and
venom gland transcriptomic analysis of Brazilian coral snakes, Micrurus altirostris
and M. corallinus, J. Proteome 74 (2011) (1795-1809).
[104] D.G. Nair, B.G. Fry, P. Alewood, P.P. Kumar, R.M. Kini, Antimicrobial activity of
omwaprin, a new member of the waprin family of snake venom proteins,
Biochem. J. 402 (2007) 93–104.
[105] R.J. Wordinger, A.F. Clark, Growth factors and neurotrophic factors as targets, in: T.
Yorio, A.F. Clark, M.B. Wax (Eds.), Ocular therapeutics: Eye on new discoveries, Ac-
ademic Press 2008, pp. 87–116.
[106] L. Aloe, Rita Levi-Montalcini: the discovery of nerve growth factor and modern
neurobiology, Trends Cell Biol. 14 (2004) 395–399.
[107] S. Cohen, R. Levi-Montalcini, Purification and properties of a nerve growth-pro-
moting factor isolated from mouse sarcoma-180, Cancer Res. 17 (1957) 15–20.
[108] S. Cohen, R. Levi-Montalcini, A nerve growth-stimulating factor isolated from
snake venom, Proc. Natl. Acad. Sci. U. S. A. 42 (1956) 571-&.
[109] S. Cohen, The stimulation of epidermal proliferation by a specific protein (EGF),
Dev. Biol. 12 (1965) 394–407.
[110] R. Ross, J. Glomset, B. Kariya, L. Harker, A platelet-dependent serum factor that
stimulates the proliferation of arterial smooth muscle cells in vitro, Proc. Natl.
Acad. Sci. U. S. A. 71 (1974) 1207–1210.
[111] T. Kostiza, J. Meier, Nerve growth factors from snake venoms: chemical properties,
mode of action and biological significance, Toxicon 34 (1996) 787–806.
[112] M.F. Lavin, S. Earl, G. Birrell, L. St. Pierre, L. Guddat, J. Jersey, P. Masci, Snake venom
nerve growth factors, in: S.P. Mackessy (Ed.), Handbook of venoms and toxins of
reptiles, CRC Press/Taylor & Francis 2010, pp. 377–391.
[113] R.H. Angeletti, Nerve growth factor from cobra venom, Proc. Natl. Acad. Sci. U. S. A.
65 (1970) 668–674.
[114] S.T. Earl, G.W. Birrell, T.P. Wallis, L.D. St Pierre, P.P. Masci, J. de Jersey, J.J. Gorman,
M.F. Lavin, Post-translational modification accounts for the presence of varied
forms of nerve growth factor in Australian elapid snake venoms, Proteomics 6
(2006) 6554–6565.
[115] D. Koh, A. Armugam, K. Jeyaseelan, Sputa nerve growth factor forms a preferable
substitute to mouse 7S-beta nerve growth factor, Biochem. J. 383 (2004) 149–158.
[116] F.L. Pearce, B.E. Banks, D.V. Banthorpe, A.R. Berry, H.S. Davies, C.A. Vernon, The iso-
lation and characterization of nerve-growth factor from the venom of Vipera
russelli, Eur. J. Biochem. 29 (1972) 417–425.
[117] R.A. Hogue-Angeletti, W.A. Frazier, J.W. Jacobs, H.D. Niall, R.A. Bradshaw, Purifica-
tion, characterization, and partial amino acid sequence of nerve growth factor
from cobra venom, Biochemistry 15 (1976) 26–34.
836 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
[118] J. Siigur, U. Arumae, T. Neuman, E. Siigur, M. Saarma, Monoclonal antibody immu-
noaffinity chromatography of the nerve growth factor from snake venoms, Comp.
Biochem. Physiol. B Biochem. Mol. Biol. 87 (1987) 329–334.
[119] L. Sanz, H.L. Gibbs, S.P. Mackessy, J.J. Calvete, Venom proteomes of closely related
Sistrurus rattlesnakes with divergent diets, J. Proteome Res. 5 (2006) 2098–2112.
[120] L.Y. Guo, J.F. Zhu, X.F. Wu, Y.C. Zhou, Cloning of a cDNA encoding a nerve growth
factor precursor from the Agkistrodon halys Pallas, Toxicon 37 (1999) 465–470.
[121] S. Kashima, A.M. Soares, P.G. Roberto, J.O. Pereira, S. Astolfi-Filho, A.O. Cintra, M.R.
Fontes, J.R. Giglio, S. de Castro Franca, cDNA sequence and molecular modeling of a
nerve growth factor from Bothrops jararacussu venomous gland, Biochimie 84
(2002) 675–680.
[122] A.V. Osipov, T.I. Terpinskaya, E.V. Kryukova, V.S. Ulaschik, L.V. Paulovets, E.A. Petrova,
E.V. Blagun, V.G. Starkov, Y.N. Utkin, Nerve growth factor from cobra venom inhibits
the growth of Ehrlich tumor in mice, Toxins (Basel) 6 (2014) 784–795.
[123] Y. Yamazaki, T. Morita, Molecular and functional diversity of vascular endothelial
growth factors, Mol. Divers. 10 (2006) 515–527.
[124] J. Boldrini-Franca, R.S. Rodrigues, F.P. Fonseca, D.L. Menaldo, F.B. Ferreira, F.
Henrique-Silva, A.M. Soares, A. Hamaguchi, V.M. Rodrigues, A.R. Otaviano, M.I.
Homsi-Brandeburgo, Crotalus durissus collilineatus venom gland transcriptome:
analysis of gene expression profile, Biochimie 91 (2009) 586–595.
[125] L. Junqueira-de-Azevedo Ide, P.L. Ho, A survey of gene expression and diversity in
the venom glands of the pitviper snake Bothrops insularis through the generation of
expressed sequence tags (ESTs), Gene 299 (2002) 279–291.
[126] R.S. Rodrigues, J. Boldrini-Franca, F.P. Fonseca, P. de la Torre, F. Henrique-Silva, L.
Sanz, J.J. Calvete, V.M. Rodrigues, Combined snake venomics and venom gland
transcriptomic analysis of Bothropoides pauloensis, J. Proteome 75 (2012)
2707–2720.
[127] I.S. Sheen, K.S. Jeng, S.C. Shih, C.R. Kao, W.H. Chang, H.Y. Wang, P.C. Wang, T.E.
Wang, L.R. Shyung, C.Z. Chen, Clinical significance of the expression of isoform
165 vascular endothelial growth factor mRNA in noncancerous liver remnants of
patients with hepatocellular carcinoma, World J. Gastroenterol. 11 (2005)
187–192.
[128] Y. Komori, T. Nikai, K. Taniguchi, K. Masuda, H. Sugihara, Vascular endothelial
growth factor VEGF-like heparin-binding protein from the venom of Vipera aspis
aspis (Aspic viper), Biochemistry 38 (1999) 11796–11803.
[129] I.L. Junqueira de Azevedo, S.H. Farsky, M.L. Oliveira, P.L. Ho, Molecular cloning and
expression of a functional snake venom vascular endothelium growth factor
(VEGF) from the Bothrops insularis pit viper. A new member of the VEGF family
of proteins, J. Biol. Chem. 276 (2001) 39836–39842.
[130] Y. Yamazaki, Y. Tokunaga, K. Takani, T. Morita, Identification of the heparin-binding
region of snake venom vascular endothelial growth factor (VEGF-F) and its
blocking of VEGF-A165, Biochemistry 44 (2005) 8858–8864.
[131] T. Kaji, C. Yamamoto, M. Oh-i, Y. Fujiwara, Y. Yamazaki, T. Morita, A.H. Plaas, T.N.
Wight, The vascular endothelial growth factor VEGF165 induces perlecan synthesis
via VEGF receptor-2 in cultured human brain microvascular endothelial cells,
Biochim. Biophys. Acta 1760 (2006) 1465–1474.
[132] T. Nieminen, P.I. Toivanen, N. Rintanen, T. Heikura, S. Jauhiainen, K.J. Airenne, K.
Alitalo, V. Marjomaki, S. Yla-Herttuala, The impact of the receptor binding profiles
of the vascular endothelial growth factors on their angiogenic features, Biochim.
Biophys. Acta 1840 (2014) 454–463.
[133] M. Simons, E. Gordon, L. Claesson-Welsh, Mechanisms and regulation of endothe-
lial VEGF receptor signalling, Nat. Rev. Mol. Cell Biol. (2016).
[134] F. Duran-Reynals, A spreading factor in certain snake venoms and its relation to
their mode of action, J. Exp. Med. 69 (1939) 69–81.
[135] A.F. Wahby, S.M. Mahdy el, H.A. El-Mezayen, W.H. Salama, A.M. Abdel-Aty, A.S.
Fahmy, Egyptian horned viper Cerastes cerastes venom hyaluronidase: purification,
partial characterization and evidence for its action as a spreading factor, Toxicon
60 (2012) 1380–1389.
[136] K.S. Girish, H.P. Mohanakumari, S. Nagaraju, B.S. Vishwanath, K. Kemparaju, Hyal-
uronidase and protease activities from Indian snake venoms: neutralization by Mi-
mosa pudica root extract, Fitoterapia 75 (2004) 378–380.
[137] K.C.F. Bordon, M.G. Perino, J.R. Giglio, E.C. Arantes, Isolation, enzymatic characteri-
zation and antiedematogenic activity of the first reported rattlesnake hyaluroni-
dase from Crotalus durissus terrificus venom, Biochimie 94 (2012) 2740–2748.
[138] J. Wohlrab, D. Wohlrab, L. Wohlrab, C. Wohlrab, A. Wohlrab, Use of hyaluronidase
for pharmacokinetic increase in bioavailability of intracutaneously applied sub-
stances, Skin Pharmacol. Physiol. 27 (2014) 276–282.
[139] C.H. Tan, S.Y. Fung, M.K. Yap, P.K. Leong, J.L. Liew, N.H. Tan, Unveiling the elusive
and exotic: Venomics of the Malayan blue coral snake (Calliophis bivirgata
flaviceps), J. Proteome 132 (2016) 1–12.
[140] G.A. Wiezel, P.K. dos Santos, F.A. Cordeiro, K.C.F. Bordon, H.S. Selistre-de-Araujo, B.
Ueberheide, E.C. Arantes, Identification of hyaluronidase and phospholipase B in
Lachesis muta rhombeata venom, Toxicon 107 (2015) 359–368.
[141] X. Xu, X.S. Wang, X.T. Xi, J. Liu, J.T. Huang, Z.X. Lu, Purification and partial character-
ization of hyaluronidase from five pace snake (Agkistrodon acutus) venom, Toxicon
20 (1982) 973–981.
[142] K. Kudo, A.T. Tu, Characterization of hyaluronidase isolated from Agkistrodon
contortrix contortrix (Southern Copperhead) venom, Arch. Biochem. Biophys. 386
(2001) 154–162.
[143] Y.H. Mahadeswaraswamy, B. Manjula, S. Devaraja, K.S. Girish, K. Kemparaju, Daboia
russelli venom hyaluronidase: purification, characterization and inhibition by beta-
3-(3-hydroxy-4-oxopyridyl) alpha-amino-propionic acid, Curr. Top. Med. Chem.
11 (2011) 2556–2565.
[144] T.C. Antunes, K.M. Yamashita, K.C. Barbaro, M. Saiki, M.L. Santoro, Comparative
analysis of newborn and adult Bothrops jararaca snake venoms, Toxicon 56
(2010) 1443–1458.
[145] G.P. Queiroz, L.A. Pessoa, F.C. Portaro, F. Furtado Mde, D.V. Tambourgi, Interspecific
variation in venom composition and toxicity of Brazilian snakes from Bothrops
genus, Toxicon 52 (2008) 842–851.
[146] C. Guerra-Duarte, J. Lopes-Peixoto, B.R. Fonseca-de-Souza, S. Stransky, D. Oliveira,
F.S. Schneider, L. Lopes-de-Souza, C. Bonilla, W. Silva, B. Tintaya, A. Yarleque, C.
Chavez-Olortegui, Partial in vitro analysis of toxic and antigenic activities of eleven
Peruvian pitviper snake venoms, Toxicon 108 (2015) 84–96.
[147] R. Shashidharamurthy, D.K. Jagadeesha, K.S. Girish, K. Kemparaju, Variations in bio-
chemical and pharmacological properties of Indian cobra (Naja naja naja) venom
due to geographical distribution, Mol. Cell. Biochem. 229 (2002) 93–101.
[148] R. Shashidharamurthy, K. Kemparaju, Region-specific neutralization of Indian
cobra (Naja naja) venom by polyclonal antibody raised against the eastern region-
al venom: A comparative study of the venoms from three different geographical
distributions, Int. Immunopharmacol. 7 (2007) 61–69.
[149] S. Pukrittayakamee, A. Nontprasert, N.J. White, D.A. Warrell, D. Bunnag, Character-
ization of a monoclonal antibody that neutralizes the hyaluronidase activity of
Russell's viper venom, Southeast Asian J. Trop. Med. Public Health 21 (1990)
231–237.
[150] K.S. Girish, K. Kemparaju, Inhibition of Naja naja venom hyaluronidase by plant-de-
rived bioactive components and polysaccharides, Biochemistry (Mosc) 70 (2005)
948–952.
[151] S.A. Khanum, S.K. Murari, B.S. Vishwanth, S. Shashikanth, Synthesis of benzoyl phe-
nyl benzoates as effective inhibitors for phospholipase A2 and hyaluronidase en-
zymes, Bioorg. Med. Chem. Lett. 15 (2005) 4100–4104.
[152] K. Sunitha, P. Suresh, M.S. Santhosh, M. Hemshekhar, R.M. Thushara, G.K. Marathe,
C. Thirunavukkarasu, K. Kemparaju, M.S. Kumar, K.S. Girish, Inhibition of hyaluron-
idase by N-acetyl cysteine and glutathione: role of thiol group in hyaluronan pro-
tection, Int. J. Biol. Macromol. 55 (2013) 39–46.
[153] M. Molander, L. Nielsen, S. Sogaard, D. Staerk, N. Ronsted, D. Diallo, K.Z. Chifundera,
J. van Staden, A.K. Jager, Hyaluronidase, phospholipase A2 and protease inhibitory
activity of plants used in traditional treatment of snakebite-induced tissue necrosis
in Mali, DR Congo and South Africa, J. Ethnopharmacol. 157 (2014) 171–180.
[154] Y. Liu, D. Staerk, M.N. Nielsen, N. Nyberg, A.K. Jager, High-resolution hyaluronidase
inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of
anti-necrosis constituents in Chinese plants used to treat snakebite, Phytochemis-
try 119 (2015) 62–69.
[155] A.N. Nanjaraj Urs, M. Yariswamy, V. Joshi, K.N. Suvilesh, M.S. Sumanth, D. Das, A.
Nataraju, B.S. Vishwanath, Local and systemic toxicity of Echis carinatus venom:
neutralization by Cassia auriculata L. leaf methanol extract, J. Nat. Med. 69 (2015)
111–122.
[156] Y.N. Utkin, A.V. Osipov, Non-lethal polypeptide components in cobra venom, Curr.
Pharm. Des. 13 (2007) 2906–2915.
[157] Y. Yamazaki, F. Hyodo, T. Morita, Wide distribution of cysteine-rich secretory pro-
teins in snake venoms: isolation and cloning of novel snake venom cysteine-rich
secretory proteins, Arch. Biochem. Biophys. 412 (2003) 133–141.
[158] Y.L. Wang, J.H. Kuo, S.C. Lee, J.S. Liu, Y.C. Hsieh, Y.T. Shih, C.J. Chen, J.J. Chiu, W.G.
Wu, Cobra CRISP functions as an inflammatory modulator via a novel Zn2+-
and heparan sulfate-dependent transcriptional regulation of endothelial cell adhe-
sion molecules, J. Biol. Chem. 285 (2010) 37872–37883.
[159] S. Lecht, R.A. Chiaverelli, J. Gerstenhaber, J.J. Calvete, P. Lazarovici, N.R. Casewell, R.
Harrison, P.I. Lelkes, C. Marcinkiewicz, Anti-angiogenic activities of snake venom
CRISP isolated from Echis carinatus sochureki, Biochim. Biophys. Acta 1850 (2015)
1169–1179.
[160] C.M. Adade, A.L. Carvalho, M.A. Tomaz, T.F. Costa, J.L. Godinho, P.A. Melo, A.P. Lima,
J.C. Rodrigues, R.B. Zingali, T. Souto-Padron, Crovirin, a snake venom cysteine-rich
secretory protein (CRISP) with promising activity against Trypanosomes and
Leishmania, PLoS Negl. Trop. Dis. 8 (2014) e3252.
[161] S.D. Aird, Taxonomic distribution and quantitative analysis of free purine and py-
rimidine nucleosides in snake venoms, Comp. Biochem. Physiol. B Biochem. Mol.
Biol. 140 (2005) 109–126.
[162] A.H. Laustsen, J.M. Gutierrez, B. Lohse, A.R. Rasmussen, J. Fernandez, C. Milbo, B.
Lomonte, Snake venomics of monocled cobra (Naja kaouthia) and investigation
of human IgG response against venom toxins, Toxicon 99 (2015) 23–35.
[163] A.H. Laustsen, B. Lomonte, B. Lohse, J. Fernández, J.M. Gutiérrez, Unveiling the na-
ture of black mamba (Dendroaspis polylepis) venom through venomics and anti-
venom immunoprofiling: Identification of key toxin targets for antivenom
development, J. Proteome 119 (2015) 126–142.
[164] C. Delezenne, H. Morel, Action catalytique des venins des serpents sur les acids
nucleiques, CR Acad. Sci. 168 (1919) 244–246.
[165] A.R. Taborda, L.C. Taborda, J.N. Williams Jr., C.A. Elvehjem, A study of the
desoxyribonuclease activity of snake venoms, J. Biol. Chem. 195 (1952) 207–213.
[166] A.R. Taborda, L.C. Taborda, J.N. Williams Jr., C.A. Elvehjem, A study of the ribonucle-
ase activity of snake venoms, J. Biol. Chem. 194 (1952) 227–233.
[167] J.G. Georgatsos, M. Laskowski Sr., Purification of an endonuclease from the venom
of Bothrops atrox, Biochemistry 1 (1962) 288–295.
[168] S. Iwanaga, T. Suzuki, Enzymes in snake venom, Snake venoms, Springer, 1979
61–158.
[169] S.P. Mackessy, Phosphodiesterases, ribonucleases and deoxyribonucleases, En-
zymes from Snake Venoms (1998) 361–404.
[170] C.L. Ownby, J. Bjarnason, A.T. Tu, Hemorrhagic toxins from rattlesnake (Crotalus
atrox) venom. Pathogenesis of hemorrhage induced by three purified toxins, Am.
J. Pathol. 93 (1978) 201–218.
[171] A.W. Bernheimer, B. Rudy, Interactions between membranes and cytolytic pep-
tides, Biochim. Biophys. Acta 864 (1986) 123–141.
[172] C.E. Nunez, Y. Angulo, B. Lomonte, Identification of the myotoxic site of the Lys49
phospholipase A2 from Agkistrodon piscivorus piscivorus snake venom: synthetic
 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membrane-
damaging activities, Toxicon 39 (2001) 1587–1594.
[173] D. Ma, A. Armugam, K. Jeyaseelan, Cytotoxic potency of cardiotoxin from Naja
sputatrix: development of a new cytolytic assay, Biochem. J. 366 (2002) 35–43.
[174] B.L. Dhananjaya, C.J.M. D'Souza, The pharmacological role of nucleotidases in snake
venoms, Cell Biochem. Funct. 28 (2010) 171–177.
[175] K. Trummal, M. Samel, A. Aaspollu, K. Tonismagi, T. Titma, J. Subbi, J. Siigur, E.
Siigur, 5'-Nucleotidase from Vipera lebetina venom, Toxicon 93 (2015) 155–163.
[176] M.C. Boffa, G.A. Boffa, Correlations between the enzymatic activities and the factors
active on blood coagulation and platelet aggregation from the venom of Vipera
aspis, Biochim. Biophys. Acta 354 (1974) 275–290.
[177] C. Ouyang, T.F. Huang, Platelet aggregation inhibitors from Agkistrodon acutus
snake venom, Toxicon 24 (1986) 1099–1106.
[178] B.L. Dhananjaya, A. Nataraju, R. Rajesh, C.D. Raghavendra Gowda, B.K. Sharath, B.S.
Vishwanath, C.J. D'Souza, Anticoagulant effect of Naja naja venom 5'-nucleotidase:
demonstration through the use of novel specific inhibitor, vanillic acid, Toxicon 48
(2006) 411–421.
[179] B. Francis, C. Seebart, I.I. Kaiser, Citrate is an endogenous inhibitor of snake venom
enzymes by metal-ion chelation, Toxicon 30 (1992) 1239–1246.
[180] E.D. Rael, Venom phosphatases and 5’-nucleotidases, Enzymes from Snake Venoms
1998, pp. 405–423.
[181] Y.H. Chen, T.B. Lo, Chemical studies of formosan cobra (Naja naja atra) venom. Part
V. Properties of 5′-nucleotidase, J. Chin. Chem. Soc. 15 (1968) 84–96.
[182] J. Dieckhoff, H. Knebel, M. Heidemann, H.G. Mannherz, An improved procedure for
purifying 5′-nucleotidase from various sources, Eur. J. Biochem. 151 (1985)
377–383.
[183] J.M. Gulland, E.M. Jackson, 5-Nucleotidase, Biochem. J. 32 (1938) 597.
[184] N.J. da Silva, S.D. Aird, Prey specificity, comparative lethality and compositional dif-
ferences of coral snake venoms, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 128
(2001) 425–456.
[185] E.A. Zeller, The formation of pyrophosphate from adenosine triphosphate in the
presence of a snake venom, Arch. Biochem. 28 (1950) 138–139.
[186] P. Caccin, P. Pellegatti, J. Fernandez, M. Vono, M. Cintra-Francischinelli, B. Lomonte,
J.M. Gutierrez, F. Di Virgilio, C. Montecucco, Why myotoxin-containing snake
venoms possess powerful nucleotidases? Biochem. Biophys. Res. Commun. 430
(2013) 1289–1293.
[187] M. Cintra-Francischinelli, P. Caccin, A. Chiavegato, P. Pizzo, G. Carmignoto, Y.
Angulo, B. Lomonte, J.M. Gutierrez, C. Montecucco, Bothrops snake myotoxins in-
duce a large efflux of ATP and potassium with spreading of cell damage and
pain, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 14140–14145.
[188] C. Gay, L. Sanz, J.J. Calvete, D. Pla, Snake venomics and antivenomics of Bothrops
diporus, a medically important pitviper in Northeastern Argentina, Toxins (Basel)
8 (2015).
[189] M. Johnson, M. Kaye, R. Hems, H. Krebs, Enzymic hydrolysis of adenosine phos-
phates by cobra venom, Biochem. J. 54 (1953) 625.
[190] T.N. Shamsi, R. Parveen, S. Fatima, Characterization, biomedical and agricultural appli-
cations of protease inhibitors: A review, Int. J. Biol. Macromol. 91 (2016) 1120–1133.
[191] L.D. Possani, B.M. Martin, A. Yatani, J. Mochca-Morales, F.Z. Zamudio, G.B. Gurrola,
A.M. Brown, Isolation and physiological characterization of taicatoxin, a complex
toxin with specific effects on calcium channels, Toxicon 30 (1992) 1343–1364.
[192] N. Willmott, P. Gaffney, P. Masci, A. Whitaker, Novel serine protease inhibitor from
the Australian brown snake, Pseudonaja textilis textilis: inhibition kinetics, Fibrino-
lysis 9 (1995) 1–8.
[193] D. Rodriguez-Ithurralde, R. Silveira, L. Barbeito, F. Dajas, Fasciculin, a powerful an-
ticholinesterase polypeptide from Dendroaspis angusticeps venom, Neurochem. Int.
5 (1983) 267–274.
[194] A.L. Harvey, Twenty years of dendrotoxins, Toxicon 39 (2001) 15–26.
[195] W.S. Yang, J. Feng, B. Wang, Z.J. Cao, W.X. Li, Y.L. Wu, Z.Y. Chen, BF9, the first func-
tionally characterized snake toxin peptide with kunitz-type protease and potassi-
um channel inhibiting properties, J. Biochem. Mol. Toxicol. 28 (2014) 76–83.
[196] J. Fernandez, J.M. Gutierrez, J.J. Calvete, L. Sanz, B. Lomonte, Characterization of a
novel snake venom component: Kazal-type inhibitor-like protein from the arbore-
al pitviper Bothriechis schlegelii, Biochimie 125 (2016) 83–90.
[197] H. Takahashi, S. Iwanaga, T. Suzuki, Isolation of a novel inhibitor of kallikrein, plas-
min and trypsin from venom of russell's viper (Vipera russelli), FEBS Lett. 27 (1972)
207-&.
[198] J.J. Calvete, C. Marcinkiewicz, L. Sanz, Snake venomics of Bitis gabonica gabonica.
Protein family composition, subunit organization of venom toxins, and character-
ization of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2, J. Proteome
Res. 6 (2007) 326–336.
[199] L. Chang, C. Chung, H.B. Huang, S. Lin, Purification and characterization of a chymo-
trypsin inhibitor from the venom of Ophiophagus hannah (king cobra), Biochem.
Biophys. Res. Commun. 283 (2001) 862–867.
[200] J. Shafqat, Z.H. Zaidi, H. Jornvall, Purification and characterization of a chymotryp-
sin kunitz inhibitor type of polypeptide from the venom of cobra (Naja naja naja),
FEBS Lett. 275 (1990) 6–8.
[201] A.K. Mukherjee, S.P. Mackessy, Pharmacological properties and pathophysiological
significance of a kunitz-type protease inhibitor (Rusvikunin-II) and its protein
complex (Rusvikunin complex) purified from Daboia russelii russelii venom,
Toxicon 89 (2014) 55–66.
[202] V. Zupunski, D. Kordis, F. Gubensek, Adaptive evolution in the snake venom kunitz/
BPTI protein family, FEBS Lett. 547 (2003) 131–136.
[203] W.M. Chou, W.H. Liu, K.C. Chen, L.S. Chang, Structure-function studies on inhibitory
activity of Bungarus multicinctus protease inhibitor-like protein on matrix
metalloprotease-2, and invasion and migration of human neuroblastoma SK-N-
SH cells, Toxicon 55 (2010) 353–360.
837
[204] A. Meta, H. Nakatake, T. Imamura, C. Nozaki, K. Sugimura, High-yield production
and characterization of biologically active recombinant aprotinin expressed in Sac-
charomyces cerevisiae, Protein Expr. Purif. 66 (2009) 22–27.
[205] A. Ritonja, B. Meloun, F. Gubensek, The primary structure of Vipera ammodytes
venom trypsin inhibitor I, Biochim. Biophys. Acta 748 (1983) 429–435.
[206] B. Gocmen, P. Heiss, D. Petras, A. Nalbantsoy, R.D. Sussmuth, Mass spectrometry
guided venom profiling and bioactivity screening of the Anatolian Meadow
Viper, Vipera anatolica, Toxicon 107 (2015) 163–174.
[207] N.H. Tan, S.Y. Fung, K.Y. Tan, M.K. Yap, C.A. Gnanathasan, C.H. Tan, Functional
venomics of the Sri Lankan russell's viper (Daboia russelii) and its toxinological
correlations, J. Proteome 128 (2015) 403–423.
[208] D. Petras, P. Heiss, R.D. Sussmuth, J.J. Calvete, Venom proteomics of Indonesian king
cobra, Ophiophagus hannah: Integrating top-down and bottom-up approaches, J.
Proteome Res. 14 (2015) 2539–2556.
[209] S.C. Wagstaff, P. Favreau, O. Cheneval, G.D. Laing, M.C. Wilkinson, R.L. Miller, R.
Stöcklin, R.A. Harrison, Molecular characterisation of endogenous snake venom me-
talloproteinase inhibitors, Biochem. Biophys. Res. Commun. 365 (2008) 650–656.
[210] D. Mora-Obando, J.A. Guerrero-Vargas, R. Prieto-Sanchez, J. Beltran, A. Rucavado,
M. Sasa, J.M. Gutierrez, S. Ayerbe, B. Lomonte, Proteomic and functional profiling
of the venom of Bothrops ayerbei from Cauca, Colombia, reveals striking interspe-
cific variation with Bothrops asper venom, J. Proteome 96 (2014) 159–172.
[211] D. Salazar-Valenzuela, D. Mora-Obando, M.L. Fernandez, A. Loaiza-Lange, H.L.
Gibbs, B. Lomonte, Proteomic and toxicological profiling of the venom of
Bothrocophias campbelli, a pitviper species from Ecuador and Colombia, Toxicon
90 (2014) 15–25.
[212] M. Kohlhoff, M.H. Borges, A. Yarleque, C. Cabezas, M. Richardson, E.F. Sanchez, Ex-
ploring the proteomes of the venoms of the Peruvian pit vipers Bothrops atrox, B.
barnetti and B. pictus, J. Proteome 75 (2012) 2181–2195.
[213] A.K. Tashima, A. Zelanis, E.S. Kitano, D. Ianzer, R.L. Melo, V. Rioli, S.S. Sant'anna, A.C.
Schenberg, A.C. Camargo, S.M. Serrano, Peptidomics of three Bothrops snake
venoms: insights into the molecular diversification of proteomes and peptidomes,
Mol. Cell. Proteomics 11 (2012) 1245–1262.
[214] I. Kregar, P. Locnikar, T. Popovic, A. Suhar, T. Lah, A. Ritonja, F. Gubensek, V. Turk, Bo-
vine intracellular cysteine proteinases, Acta Biol. Med. Ger. 40 (1981) 1433–1438.
[215] M. Brillard-Bourdet, V. Nguyen, M. Ferrer-di Martino, F. Gauthier, T. Moreau, Puri-
fication and characterization of a new cystatin inhibitor from Taiwan cobra (Naja
naja atra) venom, Biochem. J. 331 (Pt 1) (1998) 239–244.
[216] H.J. Evans, A.J. Barrett, A cystatin-like cysteine proteinase inhibitor from venom of
the African puff adder (Bitis arietans), Biochem. J. 246 (1987) 795–797.
[217] A. Ritonja, H.J. Evans, W. Machleidt, A.J. Barrett, Amino acid sequence of a cystatin
from venom of the African puff adder (Bitis arietans), Biochem. J. 246 (1987)
799–802.
[218] Y. Hamada, Y. Kiso, New directions for protease inhibitors directed drug discovery,
Biopolymers 106 (2016) 563–579.
[219] X.S. Puente, L.M. Sanchez, C.M. Overall, C. Lopez-Otin, Human and mouse prote-
ases: a comparative genomic approach, Nat. Rev. Genet. 4 (2003) 544–558.
[220] M.L. van Hoek, Antimicrobial peptides in reptiles, Pharmaceuticals (Basel) 7 (2014)
723–753.
[221] T. Ganz, Defensins: antimicrobial peptides of innate immunity, Nat. Rev. Immunol.
3 (2003) 710–720.
[222] R.P. Samy, P. Gopalakrishnakone, S.D. Satyanarayanajois, B.G. Stiles, V.T.K. Chow,
Snake venom proteins and peptides as novel antibiotics against microbial infec-
tions, Curr. Proteomics 10 (2013) 10–28.
[223] N.G. de Oliveira Junior, M.H. e Silva Cardoso, O.L. Franco, Snake venoms: attractive
antimicrobial proteinaceous compounds for therapeutic purposes, Cell. Mol. Life
Sci. 70 (2013) 4645–4658.
[224] B.A. Costa, L. Sanches, A.B. Gomide, F. Bizerra, C. Dal Mas, E.B. Oliveira, K.R. Perez, R.
Itri, N. Oguiura, M.A. Hayashi, Interaction of the rattlesnake toxin crotamine with
model membranes, J. Phys. Chem. B 118 (2014) 5471–5479.
[225] T.M. San, J. Vejayan, K. Shanmugan, H. Ibrahim, Screening antimicrobial activity of
venoms from snakes commonly found in Malaysia, J. Appl. Sci. 10 (2010)
2328–2332.
[226] A.K. Al-Asmari, R. Abbasmanthiri, N.M. Abdo Osman, Y. Siddiqui, F.A. Al-Bannah,
A.M. Al-Rawi, S.A. Al-Asmari, Assessment of the antimicrobial activity of few
Saudi Arabian snake venoms, Open Microbiol. J. 9 (2015) 18–25.
[227] M. Hakim, M. Reza, In vitro antibacterial activity of snake venom, Naja naja from
Bangladesh, Br. Biotechnol. J. 8 (2015) 1–5.
[228] B.L. Ferreira, D.O. Santos, A.L. Dos Santos, C.R. Rodrigues, C.C. de Freitas, L.M. Cabral,
H.C. Castro, Comparative analysis of viperidae venoms antibacterial profile: a short
communication for proteomics, Evid. Based Complement. Alternat. Med. 2011
(2011) 960267.
[229] R. Gennaro, M. Zanetti, Structural features and biological activities of the
cathelicidin-derived antimicrobial peptides, Biopolymers 55 (2000) 31–49.
[230] M. Zanetti, Cathelicidins, multifunctional peptides of the innate immunity, J.
Leukoc. Biol. 75 (2004) 39–48.
[231] V.M. Gomes, A.O. Carvalho, M. Da Cunha, M.N. Keller, C. Bloch Jr., P. Deolindo, E.W.
Alves, Purification and characterization of a novel peptide with antifungal activity
from Bothrops jararaca venom, Toxicon 45 (2005) 817–827.
[232] M.K. Sachidananda, S.K. Murari, D. Channe Gowda, Characterization of an antibac-
terial peptide from Indian cobra (Naja naja) venom, J. Venom. Anim. Toxins Incl.
Trop. Dis. 13 (2007) 446–461.
[233] P.G. Correa, N. Oguiura, Phylogenetic analysis of beta-defensin-like genes of
Bothrops, Crotalus and Lachesis snakes, Toxicon 69 (2013) 65–74.
[234] A.M. Siqueira, N.F. Martins, M.E. De Lima, C.R. Diniz, A. Cartier, D. Brown, B. Maigret,
A proposed 3D structure for crotamine based on homology building, molecular
simulations and circular dichroism, J. Mol. Graph. Model. 20 (2002) 389–398.
838 J. Boldrini-França et al. / Biochimica et Biophysica Acta 1861 (2017) 824–838
[235] G. Nicastro, L. Franzoni, C. de Chiara, A.C. Mancin, J.R. Giglio, A. Spisni, Solution
structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus
terrificus venom, Eur. J. Biochem. 270 (2003) 1969–1979.
[236] A.M. Torres, P.W. Kuchel, The beta-defensin-fold family of polypeptides, Toxicon
44 (2004) 581–588.
[237] L.S. Amer, B.M. Bishop, M.L. van Hoek, Antimicrobial and antibiofilm activity of
cathelicidins and short, synthetic peptides against Francisella, Biochem. Biophys.
Res. Commun. 396 (2010) 246–251.
[238] K. Fosgerau, T. Hoffmann, Peptide therapeutics: current status and future direc-
tions, Drug Discov. Today 20 (2015) 122–128.
[239] I. Schulte, H. Tammen, H. Selle, P. Schulz-Knappe, Peptides in body fluids and tis-
sues as markers of disease, Expert. Rev. Mol. Diagn. 5 (2005) 145–157.
[240] H.M. Doery, J.E. Pearson, Phospholipase B in snake venoms and bee venom,
Biochem. J. 92 (1964) 599–602.
[241] A.H. Mohamed, A. Kamel, M.H. Ayobe, Studies of phospholipase A and B activities
of Egyptian snake venoms and a scorpion toxin, Toxicon 6 (1969) 293–298.
[242] A.W. Bernheimer, S.A. Weinstein, R. Linder, Isoelectric analysis of some Australian
elapid snake venoms with special reference to phospholipase B and hemolysis,
Toxicon 24 (1986) 841–849.
[243] P. Rey-Suarez, V. Nunez, J. Fernandez, B. Lomonte, Integrative characterization of the
venom of the coral snake Micrurus dumerilii (Elapidae) from Colombia: Proteome, tox-
icity, and cross-neutralization by antivenom, J. Proteome 136 (2016) 262–273.
[244] S.T. Chatrath, A. Chapeaurouge, Q. Lin, T.K. Lim, N. Dunstan, P. Mirtschin, P.P.
Kumar, R.M. Kini, Identification of novel proteins from the venom of a cryptic
snake Drysdalia coronoides by a combined transcriptomics and proteomics ap-
proach, J. Proteome Res. 10 (2011) 739–750.
[245] D.R. Rokyta, A.R. Lemmon, M.J. Margres, K. Aronow, The venom-gland tran-
scriptome of the eastern diamondback rattlesnake (Crotalus adamanteus), BMC
Genomics 13 (2012) 312.
[246] M.J. Margres, K. Aronow, J. Loyacano, D.R. Rokyta, The venom-gland transcriptome
of the eastern coral snake (Micrurus fulvius) reveals high venom complexity in the
intragenomic evolution of venoms, BMC Genomics 14 (2013).
[247] S. Flexner, H. Noguchi, Snake venom in relation to haemolysis, bacteriolysis, and
toxicity, J. Exp. Med. 6 (1902) 277–301.
[248] J.W.W. Stephens, W. Myers, The action of cobra poison on the blood: a contribution
to the study of passive immunity, J. Pathol. Bacteriol. 5 (1898) 279–301.
[249] V. Birdsey, J. Lindorfer, H. Gewurz, Interaction of toxic venoms with the comple-
ment system, Immunology 21 (1971) 299–310.
[250] C.A. Smith, C.W. Vogel, H.J. Muller-Eberhard, Ultrastructure of cobra venom factor-
dependent C3/C5 convertase and its zymogen, factor B of human complement, J.
Biol. Chem. 257 (1982) 9879–9882.
[251] C.W. Vogel, H.J. Muller-Eberhard, Cobra venom factor: improved method for purifica-
tion and biochemical characterization, J. Immunol. Methods 73 (1984) 203–220.
[252] C.W. Vogel, H.J. Muller-Eberhard, The cobra venom factor-dependent C3
convertase of human complement. A kinetic and thermodynamic analysis of a pro-
tease acting on its natural high molecular weight substrate, J. Biol. Chem. 257
(1982) 8292–8299.
[253] D.C. Fritzinger, E.C. Petrella, M.B. Connelly, R. Bredehorst, C.W. Vogel, Primary struc-
ture of cobra complement component C3, J. Immunol. 149 (1992) 3554–3562.
[254] A.K. Abbas, A.H. Lichtman, S. Pillai, Cellular and molecular immunology, Elsevier, 2012.
[255] D.V. Tambourgi, C.W. van den Berg, Animal venoms/toxins and the complement
system, Mol. Immunol. 61 (2014) 153–162.
[256] I.L. Junqueira-de-Azevedo, C.M. Bastos, P.L. Ho, M.S. Luna, N. Yamanouye, N.R.
Casewell, Venom-related transcripts from Bothrops jararaca tissues provide novel
molecular insights into the production and evolution of snake venom, Mol. Biol.
Evol. 32 (2015) 754–766.
[257] C.A. Nicolau, P.C. Carvalho, I.L. Junqueira-de-Azevedo, A. Teixeira-Ferreira, M.
Junqueira, J. Perales, A.G. Neves-Ferreira, R.H. Valente, An in-depth snake venom
proteopeptidome characterization: Benchmarking Bothrops jararaca, J. Proteome
(2016).
[258] C.W. Vogel, D.C. Fritzinger, B.E. Hew, M. Thorne, H. Bammert, Recombinant cobra
venom factor, Mol. Immunol. 41 (2004) 191–199.
[259] C.W. Vogel, D.C. Fritzinger, Humanized cobra venom factor: experimental thera-
peutics for targeted complement activation and complement depletion, Curr.
Pharm. Des. 13 (2007) 2916–2926.
[260] J. Vejayan, T.L. Khoon, H. Ibrahim, Comparative analysis of the venom proteome of four
important Malaysian snake species, J. Venom. Anim. Toxins Incl. Trop. Dis. 20 (2014) 6.
[261] S. Li, J. Wang, X. Zhang, Y. Ren, N. Wang, K. Zhao, X. Chen, C. Zhao, X. Li, J. Shao, J.
Yin, M.B. West, N. Xu, S. Liu, Proteomic characterization of two snake venoms:
Naja naja atra and Agkistrodon halys, Biochem. J. 384 (2004) 119–127.
[262] G.W. Birrell, S.T. Earl, T.P. Wallis, P.P. Masci, J. de Jersey, J.J. Gorman, M.F. Lavin, The
diversity of bioactive proteins in Australian snake venoms, Mol. Cell. Proteomics 6
(2007) 973–986.
[263] Y.F. Pung, S.V. Kumar, N. Rajagopalan, B.G. Fry, P.P. Kumar, R.M. Kini, Ohanin, a
novel protein from king cobra venom: its cDNA and genomic organization, Gene
371 (2006) 246–256.
[264] S.D. Aird, Snake venom dipeptidyl peptidase IV: taxonomic distribution and quantita-
tive variation, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 150 (2008) 222–228.
[265] Y. Ogawa, Y. Mamura, N. Murayama, R. Yanoshita, Characterization and cDNA clon-
ing of dipeptidyl peptidase IV from the venom of Gloydius blomhoffi brevicaudus,
Comp. Biochem. Physiol. B Biochem. Mol. Biol. 145 (2006) 35–42.
[266] Y. Ogawa, M. Kanai-Azuma, Y. Akimoto, H. Kawakami, R. Yanoshita, Exosome-like
vesicles in Gloydius blomhoffii blomhoffii venom, Toxicon 51 (2008) 984–993.
[267] S.C. Yamasaki, J.S. Villarroel, J.M. Barone, L. Zambotti-Villela, P.F. Silveira, Amino-
peptidase activities, oxidative stress and renal function in Crotalus durissus
terrificus envenomation in mice, Toxicon 52 (2008) 445–454.
[268] S. Vaiyapuri, S.C. Wagstaff, K.A. Watson, R.A. Harrison, J.M. Gibbins, E.G.
Hutchinson, Purification and functional characterisation of rhiminopeptidase A, a
novel aminopeptidase from the venom of Bitis gabonica rhinoceros, PLoS Negl.
Trop. Dis. 4 (2010) e796.
[269] S. Zini, M.C. Fournie-Zaluski, E. Chauvel, B.P. Roques, P. Corvol, C. Llorens-Cortes,
Identification of metabolic pathways of brain angiotensin II and III using specific
aminopeptidase inhibitors: predominant role of angiotensin III in the control of va-
sopressin release, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 11968–11973.
[270] I.M. Francischetti, V. My-Pham, J. Harrison, M.K. Garfield, J.M. Ribeiro, Bitis gabonica
(Gaboon viper) snake venom gland: toward a catalog for the full-length transcripts
(cDNA) and proteins, Gene 337 (2004) 55–69.
[271] L. Qinghua, Z. Xiaowei, Y. Wei, L. Chenji, H. Yijun, Q. Pengxin, S. Xingwen, H.
Songnian, Y. Guangmei, A catalog for transcripts in the venom gland of the
Agkistrodon acutus: identification of the toxins potentially involved in coagulopa-
thy, Biochem. Biophys. Res. Commun. 341 (2006) 522–531.
[272] H. Ichijo, U. Hellman, C. Wernstedt, L.J. Gonez, L. Claesson-Welsh, C.H. Heldin, K.
Miyazono, Molecular cloning and characterization of ficolin, a multimeric protein
with fibrinogen- and collagen-like domains, J. Biol. Chem. 268 (1993) 14505–14513.
[273] Y. Kakinuma, Y. Endo, M. Takahashi, M. Nakata, M. Matsushita, S. Takenoshita, T.
Fujita, Molecular cloning and characterization of novel ficolins from Xenopus laevis,
Immunogenetics 55 (2003) 29–37.
[274] G. OmPraba, A. Chapeaurouge, R. Doley, K.R. Devi, P. Padmanaban, C. Venkatraman,
D. Velmurugan, Q. Lin, R.M. Kini, Identification of a novel family of snake venom
proteins veficolins from Cerberus rynchops using a venom gland transcriptomics
and proteomics approach, J. Proteome Res. 9 (2010) 1882–1893.
[275] A.T. Ching, M.M. Rocha, A.F. Paes Leme, D.C. Pimenta, M. de Fátima D Furtado, S.M.
Serrano, P.L. Ho, I.L. Junqueira-de-Azevedo, Some aspects of the venom proteome
of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom)
gland transcriptome, FEBS Lett. 580 (2006) 4417–4422.
[276] L. St Pierre, R. Woods, S. Earl, P.P. Masci, M.F. Lavin, Identification and analysis of
venom gland-specific genes from the coastal taipan (Oxyuranus scutellatus) and re-
lated species, Cell. Mol. Life Sci. 62 (2005) 2679–2693.
[277] M.J. Margres, J.J. McGivern, K.P. Wray, M. Seavy, K. Calvin, D.R. Rokyta, Linking the
transcriptome and proteome to characterize the venom of the eastern diamond-
back rattlesnake (Crotalus adamanteus), J. Proteome 96 (2014) 145–158.
[278] C.R. Diniz, J.M. Gonçalves, Separation of biologically active components from scor-
pion venoms by zone electrophoresis, Biochim. Biophys. Acta 41 (1960) 470–477.
[279] N.R. Casewell, R.A. Harrison, W. Wüster, S.C. Wagstaff, Comparative venom gland
transcriptome surveys of the saw-scaled vipers (Viperidae: Echis) reveal substan-
tial intra-family gene diversity and novel venom transcripts, BMC Genomics 10
(2009) 564.
[280] S.C. Wagstaff, R.A. Harrison, Venom gland EST analysis of the saw-scaled viper,
Echis ocellatus, reveals novel alpha9beta1 integrin-binding motifs in venom metal-
loproteinases and a new group of putative toxins, renin-like aspartic proteases,
Gene 377 (2006) 21–32.
[281] A. Chapeaurouge, M.A. Reza, S.P. Mackessy, P.C. Carvalho, R.H. Valente, A. Teixeira-
Ferreira, J. Perales, Q. Lin, R.M. Kini, Interrogating the venom of the viperid snake
Sistrurus catenatus edwardsii by a combined approach of electrospray and MALDI
mass spectrometry, PLoS One 10 (2015) e0092091.
[282] D. Georgieva, J. Seifert, M. Öhler, M. von Bergen, P. Spencer, R.K. Arni, N. Genov, C.
Betzel, Pseudechis australis venomics: adaptation for a defense against microbial path-
ogens and recruitment of body transferrin, J. Proteome Res. 10 (2011) 2440–2464.
[283] I.L. Junqueira-de-Azevedo, T. Pertinhez, A. Spisni, F.R. Carreno, C.S. Farah, P.L. Ho, Clon-
ing and expression of calglandulin, a new EF-hand protein from the venom glands of
Bothrops insularis snake in E. coli, Biochim. Biophys. Acta 1648 (2003) 90–98.
[284] J.J. Calvete, P. Juarez, L. Sanz, Snake venomics. Strategy and applications, J. Mass
Spectrom. 42 (2007) 1405–1414.
[285] B.G. Fry, From genome to "venome": molecular origin and evolution of the snake
venom proteome inferred from phylogenetic analysis of toxin sequences and relat-
ed body proteins, Genome Res. 15 (2005) 403–420.
[286] R. Schoni, The use of snake venom-derived compounds for new functional diag-
nostic test kits in the field of haemostasis, Pathophysiol. Haemost. Thromb. 34
(2005) 234–240.
[287] D.C.I. Koh, A. Armugam, K. Jeyaseelan, Snake venom components and their applica-
tions in biomedicine, Cell. Mol. Life Sci. 63 (2006) 3030–3041.
[288] Y.S. Chan, R.C. Cheung, L. Xia, J.H. Wong, T.B. Ng, W.Y. Chan, Snake venom toxins:
toxicity and medicinal applications, Appl. Microbiol. Biotechnol. 100 (2016)
6165–6181.
[289] A.L. Harvey, Toxins and drug discovery, Toxicon 92 (2014) 193–200.
[290] A.D. Hargreaves, M.T. Swain, M.J. Hegarty, D.W. Logan, J.F. Mulley, Restriction and
recruitment-gene duplication and the origin and evolution of snake venom toxins,
Genome Biol. Evol. 6 (2014) 2088–2095.
[291] J. Reyes-Velasco, D.C. Card, A.L. Andrew, K.J. Shaney, R.H. Adams, D.R. Schield, N.R.
Casewell, S.P. Mackessy, T.A. Castoe, Expression of venom gene homologs in di-
verse python tissues suggests a new model for the evolution of snake venom,
Mol. Biol. Evol. 32 (2015) 173–183.
[292] V.J. Lynch, Inventing an arsenal: adaptive evolution and neofunctionalization of
snake venom phospholipase A2 genes, BMC Evol. Biol. 7 (2007) 2.
[293] B.G. Fry, W. Wuster, R.M. Kini, V. Brusic, A. Khan, D. Venkataraman, A.P. Rooney,
Molecular evolution and phylogeny of elapid snake venom three-finger toxins, J.
Mol. Evol. 57 (2003) 110–129.
[294] N.R. Casewell, W. Wuster, F.J. Vonk, R.A. Harrison, B.G. Fry, Complex cocktails: the
evolutionary novelty of venoms, Trends Ecol. Evol. 28 (2013) 219–229.
[295] N.R. Casewell, S.C. Wagstaff, R.A. Harrison, C. Renjifo, W. Wuster, Domain loss facil-
itates accelerated evolution and neofunctionalization of duplicate snake venom
metalloproteinase toxin genes, Mol. Biol. Evol. 28 (2011) 2637–2649.